Monday June 14th
08:30 - 09:00 Introduction
09:00 - 13:00 Bacteriology and Genetics
09:00 - 10:00
Transformation
Chair : Akira TAMURA & Cecilia MORON
09:00 - 09:20
Rickettsia
09:20 - 09:40
Genetic transformation of Coxiella burnetii
origins, vectors, and recombinations
09:40 - 10:00
10:00 - 10:40
David WOOD (USA)
Herbert THOMPSON (USA)
Genetic manipulation of Bartonella
Genes and Genome
Michael MINNICK (USA)
Chair : Michael MINNICK & Daniel PARZY
10:00 - 10:20
Sequencing of the Coxiella burnetii plasmids
10:20 - 10:40
Bartonella-endothelial cell interaction
Hermann WILLEMS (Germany)
10:40 - 11:20
Coffee break and visit of Poster Session 1
11:20 - 12:00
Genetic diversity
Chair : Herbert THOMPSON & Debra SWEGER
11:20 - 11:40
Genetic diversity of Rickettsia
11:40 - 12:00
Genetic diversity of 0rientia tsutsugamushi
12:00 - 12:50
12:00 - 12:10
Burt ANDERSON (USA)
Slide Session 1
Véronique ROUX (France)
Akira TAMURA (Japan)
Chair : Herbert WINKLER & Laura HENDRIX
A tail-specific protease from Bartonella quintana
may be involved in hemin acquisition
Michael MINNICK (USA)
12:10 - 12:20
Molecular analysis of strain-specific protective antigen
and GROEL homologue protein of Ehrlichia risticii
Sukanta DUTTA (USA)
12:20 - 12:30
Evaluation of protective capacity of Cowdria ruminantium
genes encoding immunogenic proteins
by DNA immunization
Suman MAHAN (Zimbabwe)
12:30 - 12:40
Differential expression of transcriptionnal and translational
elements by Coxiella burnetii life cycle stages
Rekha SESHADRI (USA)
12:40 - 12:50
Cloning and characterization of a Heat Shock
sigma 32 homolog in Coxiella burnetii
13:00 - 14:30 Lunch (IMTSSA)
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Janakiram SESHU (USA)
14:30 - 16:50 Physiopathology and Immunity
14:30 - 17:10
Lectures
Chair : Tsuneo UCHIYAMA & Mustapha AKKOYUNLU
14:30 - 14:50
Physiology of rickettsial infection
14:50 - 15:10
Immunity in Q fever
15:10 - 15:30
Biology of Coxiella burnetii
James SAMUEL (USA)
15:30 - 15:50
Intracellular motility of Rickettsia
Robert HEINZEN (USA)
15:50 - 16:10
Immunopathogenesis of Boutonneuse Fever
16:10 - 16:30
Rickettsia rickettsii infection of human endothelial cells:Oxidative
injury and reorganisation of the cytoskeleton
Marina EREMEEVA (USA)
16:30 - 16:50
Lispopolysacharides from virulent and low virulent
phase of Coxiella burnetii
16:50 - 17:10
Herbert WINKLER (USA)
Jean-Louis MEGE (France)
Intraerythrocytic infection of Bartonella
Enrico CILLARI (Sicily)
Rudolf TOMAN (Slovakia)
Christoph DEHIO (Germany)
17:10 - 17:40
Coffee break and visit of Poster Session 2
17:40 - 19:50
Slide Session 2
17:40 - 17:50
Chair : Xue-Jie YU & Suman MAHAN
Intracellular anti-rickettsial mechanisms of chemokineand cytokine-activated human macrophages
and hepatocytes
David WALKER (USA)
17:50 - 18:00
The surface protein antigen of Rickettsia typhi: in vitro and
in vivo immunogenicity and protective efficacy in mice Gregory DASCH (USA)
18:00 - 18:10
Persistence of Coxiella burnetii infection
18:10 - 18:20
Experimental infection of cats and dogs with Bartonella
isolated from domestic and wild carnivores
Bruno CHOMEL (USA)
18:20 - 18:30
Molecular heterogeneity of 28 kDa surface antigen multigen
locus of Ehrlichia chaffeensis isolates suggests that it plays
a role in immune evasion
Roman REDDY (USA)
18:30 - 18:40
Mechanisms of immunity to Ehrlichia chaffeensis in mice Hui-Min FENG (USA)
18:40 - 18:50
Characterization of the genus-common Outer Membrane
Proteins in Ehrlichia
Ray HARRIS (Australia)
Xue-Jie YU (USA)
18:50 - 19:00
Interferon gamma dominates the early cytokine response
to murine infection with the agent of human
granulocytic ehrlichiosis
Mustafa AKKOYUNLU (USA)
19:00 - 19:10
A feline model of granulocytic ehrlichiosis
infection with AIDS
Janet FOLEY (USA)
19:10 - 19:20
Cowdria MAP1 protein sequence similarity clustering
and cross protection among isolates
Maria ALLSOPP (South Africa)
19:20 - 19:30
Optimization of the MAP1 DNA vaccine for sheep
19:30 - 19:40
The role of T cells in immunity to Cowdria
ruminantium infection
19:40 - 19:50
Importance of antigenic variation in persistence
of the ehrlichia Anaplasma marginale
Michael BOWIE (USA)
Suman MAHAN (Zimbabwe)
Guy PALMER (USA)
10:00 - 20:00 Non Stop Visit of Poster sessions 1 and 2
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Tuesday June 15th
08:00 - 12:40 Emerging infections
08:00 - 10:10
Emerging rickettsioses throughout the world (Part 1)
Chair : Irina TARASEVICH & Marcio GALVAO
08:00 - 08:20
Emerging rickettsioses
Didier RAOULT (France)
08:20 - 08:40
Emerging infectious diseases in the Americas
08:40 - 09:00
Rickettsioses in Australia
09:00 - 09:20
Rickettsioses in Japan
Fumihiko MAHARA (Japan)
09:20 - 09:40
New rickettsiosis in Russia
Irina TARASEVICH (Russia)
09:40 - 10:00
Q fever - Current concept
David WALKER (USA)
Stephen GRAVES (Australia)
Ian KAZAR (Slovakia)
10:00 - 10:40
Coffee break and visit of Poster Session 3
10:40 - 11:40
Emerging rickettsioses throughout the world (Part 2)
Chair : Johan BAKKEN & Stanka LOTRIC-FURLAN
10:40 - 11:00
Ehrlichiosis in Europe
11:00 - 11:20
HGE in North America : understanding clinical
disease and pathogenesis with animal models
11:20 - 11:40
11:40 - 13:00
11:40 - 11:50
Philippe BROUQUI (France)
Monocytic Ehrlichiosis in the United States :
well are we counting the cases ?
Slide Session 3
Stephen DUMLER (USA)
James CHILDS (USA)
Chair : Fumihiko MAHARA & Anne-Marie PRETORIUS
Tick-borne lymphadenopathy (TIBOLA):
a Rickettsia slovaca infection
Andras LAKOS (Hungary)
11:50 - 12:00
Rickettsia felis : the etiologic agent of a case
of rickettsiosis in the Yucatan
Jorge ZAVALA-VELASQUEZ (USA)
12:00 - 12:10
Etiology of febrile illnesses after a tick bite
in Slovenian patients with leukopenia
and/or thrombocytopenia
Stanka LOTRIC-FURLAN (Slovenia)
12:10 - 12:20
Granulocytic Ehrlichiae infections in humans,
ticks and wild mammals in western Switzerland
Jorge LIZ (Switzerland)
Limits to infection with the Human Granulocytic
Ehrlichiosis agent
Thomas MATHER (USA)
12:20 - 12:30
12:30 - 12:40
Temporal and spacial dynamics of Ehrlichia
phagocytophila transmission in northeastern USA
Durland FISH (USA)
13:00 - 14:30 Lunch (IMTSSA)
14:30 - 16:50 Diagnosis and Treatment of Rickettsioses
-3-
Lectures
Chair : James CHILDS & Neus CARDENOSA
14:30 - 14:50
Serologic diagnosis of Rickettsiosis
Bernard LA SCOLA (France)
14:50 - 15:10
Serologic diagnosis of Bartonellosis
Russel REGNERY (USA)
15:10 - 15:30
Molecular diagnostic of ehrlichioses
and rickettsioses
Robert MASSUNG (USA)
Developing new molecular tools for
universal diagnosis of rickettsiae :
the RPOB encoding gene
Michel DRANCOURT (France)
15:30 - 15:50
15:50 - 16:10
Clinical and therapeutical approach to MSF
16:10 - 16:30
In vitro susceptibility of rickettsiae to antibiotics
16:30 - 16:50
Vaccine prophylaxis of Q fever
16:50 - 17:20
Ferran SEGURA-PORTA (Spain)
Max MAURIN (France)
Barrie MARMION (Australia)
Coffee break and visit of Poster Session 4
17:20 - 19:00 Epidemiology, Diagnosis and Treatment
Slide Session 4
Chair : Amel LETAIEF & Stephen GRAVES
17:20 - 17:30
Hidden mortality attributable to Rocky Mountain Spotted fever :
immunohistochemical detection of fatal,
serologically unconfirmed disease
Christopher PADDOCK (USA)
17:30 - 17:40
Laboratory diagnosis of rickettsial diseases
in Queensland
Camer TAYLOR (Australia)
17:40 - 17:50
Coxiella burnetii in ticks, goats, sheep and humans
in Cyprus : detection, isolation and molecular
identification of six strains
Iannis TSELENTIS (Greece)
17:50 - 18:00
Seroepidemiology of bartonellosis in
Poland
Stanislawa TYLEWSKA-WIERZBANOWSKA (Poland)
18:00 - 18:10
Human ehrlichiosis surveillance
in the United States
18:10 - 18:20
18:20 - 18:30
18:10 - 18:20
18:30 - 18:40
18:40 - 18:50
Jennifer Mc QUISTON (USA)
Influence of occupation, pre-existing illness,
and chronic medication use on the severity of illness
of human granulocytic ehrlichiosis
Johan BAKKEN (USA)
Immunodiagnosis of human granulocytic ehrlichiosis
using a recombinant HGE-44-based ELISA
Jacob IJDO (USA)
Human Granulocytic Ehrlichiosis
in Scandinavia
Bartonella bacilliformis in the Sacred Valley
of the Incas, Cusco, Peru, 1998
Anneli BJOERSDORFF (Sweden)
Palmira VENTOSILLA (PERU)
Improved sensitivity of PCR for HGE using
EPANK gene of E. phagocytophila group ehrlichiae
Jennifer WALLS (USA)
19:00 - 20:00 Plenary session of the ASR
10:00 - 20:00 Non Stop Visit of Poster sessions 3 and 4
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Wednesday June 16th
08:00 - 12:45 Rickettsioses in Animals, Hosts and Vectors
08:30 - 10:15
Rickettsioses in animals
Chair : Anneli BJOERSDORFF & Bernard DAVOUST
08:30 - 09:15
Ehrlichiae of veterinary importance
09:15 - 09:35
Recent developments in the pathogenesis
of tick-borne fever
09:35 - 09:55
09:55 - 10:15
Yasuko RIKIHISA (USA)
Zerai WOLDEHIWET (UK)
Recent developments in research
on Anaplasma marginale
Anthony BARBET (USA)
Cowdria ruminantium : recent developments
in diagnostic methods, molecular
characterization and vaccines
Frans JONGEJAN (Netherlands)
10:15 - 10:45
Coffee break and visit of Poster Session 5
10:45 - 11:45
Host - Vector - Parasite
Chair : Gladia MACRI & Henk BRAIG
10:45 - 11:05
Ticks and Ehrlichia, Babesia and Borrelia
11:05 - 11:25
Lice, Rickettsia prowazekii and Bartonella quintana
11:25 - 11:45
Ticks and Rickettsiae in Portugal
11:45 - 12:45
11:45 - 11:55
Slide Session 5
Sam TELFORD (USA)
Elena RYDKINA (Russia)
Fatima BACELLAR (Portugal)
Chair : I. KURHANOVA & Joseph BUNNEL
Validation and application of the Cowdria
ruminantium-specific pCS20 Tick PCR assay
Suman MAHAN (Zimbabwe)
11:55 - 12:05
A chemically defined medium for the in vitro
cultivation of Cowdria ruminantium
Erich ZWEYGARTH (South Africa)
12:05 - 12:15
Spotted fever group Rickettsiae in Ixodes
ricinus ticks in Slovenia
Tatjana AVSIC-ZUPAN (Slovenia)
12:15 - 12:25
The many faces of Wolbachia
12:25 - 12:35
Strain structure of the ehrlichia Anaplasma
marginale within endemic herds and association
with transmission
12:35 - 12:45
Henk BRAIG (UK)
Terry Mc ELWAIN (USA)
Rickettsia felis : Identification and molecular
characterization in fleas (Ctenocephalides felis)
in Yucatan
Jorge ZAVALA-VELASQUEZ (USA)
12:45 - 14:15 Lunch (IMTSSA)
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14:15 - 16:45 Taxonomy
14:15 - 15:15
Special lecture
Comparison of complete bacterial genome :
a new approach for bacterial phylogeny
Jean-Michel CLAVERY (France)
15:15 - 16:45
Taxonomic comittee
16:45 - 17:15
Coffee break and visit of Poster Session 6
17:00 - 18:00 Plenary session of the EUWOG
10:00 - 18:30 Non Stop Visit of Poster sessions 5 and 6
19:00
Farwell Party
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Abstracts
The full text of the lectures is given in the book
"Rickettsiae and Rickettsial Diseases at the Turn of the Third Century"
These abstracts are therefore not reproduced here.
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Monday June 14th
Poster session 1
Bacteriology and Genetics
Contact-dependent hemolytic activity distinct from deforming
activity by Bartonella bacilliformis
L. Hendrix
Texas A&M University Health Science Center, College Station, TX, USA
Although Bartonella bacilliformis causes a severe anemia in humans, an ability
of the organism to lyse erythrocytes has previously never been demonstrated. This
study reports the hemolytic activity by B. bacilliformis. The activity was not found in
culture supernatants and was more reliably detected when B. bacilliformis cells were
centrifuged onto erythrocytes before incubation. Inhibitor studies with proteinase K
suggested the hemolysin was a Bartonella protein. A concentration of factors
released by B. bacilliformis containing deformin, a B. bacilliformis protein able to
induce pits and invaginations in erythrocyte membranes, had some ability to lyse
erythrocytes. Inhibition of deformations caused by B. bacilliformis cells with the
erythrocyte ATPase inhibitor, vanadate, did not have any effect on hemolytic activity.
This suggests hemolytic activity and deforming activity are attributable to two different
B. bacilliformis proteins. A genetic system enabling the selection of mutants for these
activities is being developed, using a vector containing a constituitively expressed gfp
gene, the sacB gene and the Tn5 minitransposon. B. bacilliformis transformed with a
vector containing the sacB gene were no longer able to grow on 5% sucrose, but
when antibiotic selection required to maintain the plasmid was removed, the ability of
B. bacilliformis to grow on 5% sucrose was restored. The use of the sacB
counterselectable marker system should increase the rate of isolation of transposon
mutants, which can then be screened in assays developed to detect these activities.
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29
Role of major surface antigens of Rickettsia japonica in the
attachment to host cells
46
Tsuneo Uchiyama
Department of Virology, School of Medicine, The University of Tokushima, 3-18-15
Kuramoto-cho, Tokushima 770-8503, Japan
Role of major surface antigens of Rickettsia japonica in the attachment of the
organisms to the host cells was examined by using monospecific antibodies (MsAbs)
and recombinant fusion proteins. Recombinant major outer membrane proteins, rOmp
A and rOmp B, of R. japonica were expressed as inclusion bodies in Escherichia coli.
The insoluble proteins were solubilized in a buffer containing urea and purified by
means of affinity column chromatography. Refolding of the proteins to have soluble
conformations was achieved by dialysis of the proteins in the presence of chaperonin
GroE. These soluble proteins demonstrated competitive inhibition of attachement of
the rickettsiae to the host Vero cells to some extent. F(ab')2 fragment of the antibodies
to live rickettsiae and MsAbs against native rOmp A and rOmp B also demonstrated
inhibition of the attachement. MsAbs against the completely denatured rOmp A and
rOmp B demonstrated almost no inhibition. These results suggest that these proteins
have some role in the attachement of the organisms and that native conformations are
important for these proteins to function.
Nucleotide metabolism in variants of Coxiella burnetii Nine-Mile
strain.
Jeffrey D. Miller & Herbert A. Thompson
West Virginia University Heath Sciences Center. Department of Microbiology and
Immunology, Morgantown, WV 26506 USA
The capabilities for nucleotide metabolism in Coxiella burnetii and the
organism's degree of dependence on the host for purines and pyrimidines are
incompletely defined. This host-parasite interaction may also have an effect upon
macromolecular synthesis in the organism, including the formation of complete
lipopolysaccharide (LPS) on the outer membrane. We have investigated some effects
of nucleoside and nucleotide supplementation on C. burnetii growth in tissue culture.
Supplementation with cytidine has shown significant stimulatory effects on some
variants of Coxiella Nine-Mile strain when studied during growth in Baby Hamster
Kidney cells. Ongoing studies have therefore been designed to utilize both genetic
and biochemical techniques to characterize the uptake, metabolism, and incorporation
of nucleosides and nucleoside precursors. Genetic studies incorporated PCR and
Southern Blot Hybridization technology to isolate specific gene DNA of interest for
sequencing. Biochemical studies included the use of radiolabeled tracers in
combination with Thin-layer Chromatography (TLC), High Performance Liquid
Chromatography (HPLC), and UV/Visible spectrophotometry to isolate gene function
and activity. This study focused on the presence or absence of CTP Synthetase
activity within C. burnetii Nine-Mile variants, and the occurrence and activity of a CTP
synthetase-like gene.
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53
Sequence analysis of rOmpA gene fragment of several new
strains of spotted fever group rickettsiae isolated from various
sources in china
56
Chen Min, Zhang Jian-zhi, Bi De-zeng
Departrnent of Rickettsiology, Insitute of Epiderniology and Microbiology, Chinese
Academy of Preventive Medicine,102206 Beijing;
The primer, Rr 90-4442p-5664n was used to amplify the rOmpA gene fragment
of several Chinese strains of spotted fever group rickettsiae(SFGR). The PCR
products were sequenced directly by Sanger’s dideoxy method. The obtained
nucleotide and putative amino acid sequences were compared with each other This
comparison showed that the homology of isolate BJ-90(isolated from Dermacentor
sinicus) with Ha-91 (isolated from Hyalomma asiaticum), HLJ-054 (isolated from
Dermacentor silvarum)and HL-93 (isolated fiorn Haemaphysalis concinna) were
98.66%, 97.1% and 97.08% in nucleotide and 99.00%, 94.25% and 95.00% in
putative amino acid respectively. In relation to Ha-91, tu homology with HLJ-054 and
HL-93 was 97.33% and 97.l6% In nucleotide and 94.75% and 95.00% In putative
aminO acid. HLJ-054 showed higher homology with HL-93 than others (99.41% in
nucleotide and 98.75% in putative amino acid). We assume that not only the isolates
BJ-90 and Ha-91 but also HL-93 and HLJ-054 are closely related, the latter two may
represent new, unique members of SFGR.
Luciferase as a genetic marker for the detection of transformation
in Coxiella burnetii
Michelle L. Suhan1, Aaron Ruhland1, Michael Vodkin2, and Herbert A.
Thompson1.
1 West Virginia University, Department of Microbiology, Morgantown, WV 26506 USA
2 Illinois Natural History Survey, University of Illinois, Urbana, USA
C. burnetii can be electro-transformed to ampicillin resistance by using the
plasmid, pSKO(+)1000 which contains the C. burnetii autonomous replication
sequence (ars) on a 5.8-kb chromosomal fragment cloned into an Escherichia coli
ColE1 plasmid encoding for beta-lactamase. Difficulties associated with genetic
transformation of this obligate intracellular parasite, including a slow growth rate and
persistence of organisms in long term antibiotic selection, necessitates improved,
quicker detection of transformants. This laboratory has investigated the use of
luciferase as a marker to detect electro-transformed C. burnetii. C. burnetii were
electroporated with plasmids encoding for luciferase from either a heat shock
promoter from the C. burnetii htpA,B operon, or a housekeeping promoter from the C.
burnetii citrate synthase gene, gltA. Constructs also contained the E. coli ColE1
replicon, beta-lactamase gene, and either the 5.8-kb C. burnetii ars or the 568-bp
minimal ars. Following electroporation cells were incubated in an acid activation
medium to stimulate macromolecular synthesis. However, luciferase activity was not
detected in extracts prepared from acid activated cells. To determine whether the
luciferase gene from these plasmids survived in the bacteria, electro-transformed C.
burnetii grown in BHK cells, cultivated in the presence of ampicillin, were examined by
PCR and Southern blot analysis. These results suggest that luciferase will not
function as a stable marker for genetic transformation of C. burnetii.
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58
Molecular characterization of a new 28-kilodalton protein gene and
a multigene locus encoding five homologous 28-kilodalton
immunodominant outer membrane proteins of Ehrlichia canis.
60
Jere W. McBride*, Xue-jie Yu, and David H. Walker.
Department of Pathology, University of Texas Medical Branch, WHO Collaborating
Center for Tropical Diseases, Galveston, TX 77555-0609
Recently four major immunodominant outer membrane 28-kilodalton protein
genes in Ehrlichia canis, and six homologous genes in Ehrlichia chaffeensis have
been identified and sequenced. Using PCR to amplify p28 genes of E. canis, a
previously unsequenced region of one E. canis p28 gene (Eca28SA2)was completed,
and a new 28-kilodalton protein gene in E. canis (ECa28SA3) was identified and the
complete sequence determined. Sequence analysis of ECa28SA2 revealed a 849-bp
ORF encoding a 283 amino acid protein, and the ORF of ECa28SA3, was 843-bp in
length encoding a 281 amino acid protein. PCR amplification using primers specific
for p28 intergenic noncoding regions linked two previously separate loci, resulting in a
single locus (5.65-kb) containing all five p28 genes. The five p28 proteins were
predicted to have signal peptides resulting in mature proteins, and amino acid
homology ranging from 51 to 71%. Nucleic acid analysis of one p28 genes (ECa28-1)
demonstrated complete conservation among 7 different isolates of E. canis. Analysis
of intergenic regions revealed hypothetical promoter regions for each gene,
suggesting that these genes may be independently and differentially expressed.
Intergenic noncoding regions ranged in size from 299 to 355-bp, and were 48 to 71%
homologous.
The identification and characterization of a previously
Undiscovered rOmpA encoding gene in Rickettsia felis.
Donald H. Bouyer1, Patricia Crocquet-Valdes1, John Stenos2, Lane Foil3 and
David H. Walker1.
Department of Pathology, University of Texas Medical Branch, Galveston, Texas
77550-06091; Geelong Hospital, Geelong, Australia2; Department of Entomology, LSU
Agricultural Center, Baton Rouge, LA 70803-17103.
A recently identified rickettsiae, Rickettsia felis, has been included in the
typhus group rickettsiae on the basis of serological means. R. felis has been found to
react with antisera to R. typhi and PCR/RFLP analysis of the conserved 17 kDa gene
also places R. felis within the typhus group. Genetic analysis of the 16s rRNA gene
and the citrate synthase gene place R. felis in the same clade as R. akari and R.
australis, members of the spotted fever group. Previous attempts to classify R. felis
as a spotted fever group rickettsiae by PCR amplification of the rOmpA gene using
primers Rr.190.70 and Rr.190.602n have been unsuccessful
In an effort to determine in which group that R. felis should be included, we
attempted to PCR amplify the rOmpA gene from Ctenocepalides felis cat fleas
containing R. felis using different primers and amplification conditions. In this study,
we report the successful sequencing and characterization of the rOmpA gene from
Rickettsia felis. The approximately 6 kb gene has regions that have high percentages
of similarity to R. akari, R. australis, and R. rickettsii. The repeat region of the R. felis
rOmpA gene is unique in that it contains only 5 of the repeating units. These results
indicate that Rickettsia felis does contain a previously undiscovered gene encoding
rOmpA and that its present classification as a member of the typhus group rickettsiae
may need to be reviewed.
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62
Lipopolysaccharides in phase variation of Coxiella burnetii
Ftácek P, Škultéty L', and Toman R
Institute of Virology Slovak Academy of Sciences. Dûbravská cesta
Bratislava, Slovak Republic
66
9. 842 46.
Coxiella burnetii, the causative agent of Q fever, may undergo a transition from
virulent phase I to avirulent phase II by serial passage in the yolk sacs of
embryonated eggs. Lipopolysaccharides (LPSs) isolated from Coxiella burnetii phase
I cells contain mainly long carbohydrate chains ( O-specific ) whereas in Coxiella
burnetii phase Il cells prevail short LPS molecules. Phase variation of Coxiella burnetii
was often compared with smooth-to-rough (S  R) mutation described in many
Enterobacteriaceae as for the changes in its LPS composition and structure, but no
detailed study has been done in this direction.
In our study, the LPSs isolated from the purified cells of various egg passages
(EP) were fractionated by steric-exclusion chromatography into the three major
groups : high, intermediate, and low molecular weight fractions. The fractions were
analyzed for chemical composition by various physico-chemical methods. The
molecular heterogeneity and size distribution of the individual LPSs was investigated
by SDS-PAGE and TLC.
The results revealed the following (i) Coxiella burnetii is capable of
biosynthesizing several subclasses of LPS molecules, (ii) the three LPS subclasses
were isolated from all investigated Coxiella burnetii cells of different EP (3 to 90), (iii)
the distribution ratio of these subclasses is characteristic for Coxiella burnetii cells in
the respective EP, and (iv) progress in EP has increased the content of low molecular
weight portion of LPS in the overall LPS size population. Thus, compositional and
structural changes in LPS during Coxiella burnetii phase variation seem to differ from
those observed in S R mutation of Enterobacteriaceae.
Codon usage in Coxiella burnetii
S. Lautenschlaeger, H. Willems, C. Jaeger and G. Baljer
Codon usage (CU) patterns and base composition were analyzed for 49
Coxiella (C.) burnetii chromosomal genes, the C. burnetii plasmids QpH1, QpRS and
QpDV in total and phage lambda DNA, respectively. Synonymous codon usage
(SCU), relative synonymous codon usage (RSCU) and rare codons were determined.
CU patterns across genes were analyzed by Nc-plots, a plot of SCU bias vs. G + C
content in the third codon position. The degree of SCU bias is due to codon
preference in genes and in unicellular organisms highly correlated with the level of
gene expression. CU patterns are roughly similar among C. burnetii plasmid genes
but differ strongly to C. burnetii chromosomal and bacteriophage lambda genes. The
CU patterns of plasmids revealed no relatedness to phage DNA although phages are
discussed as putative plasmid ancestors. The number of rare codons in C. burnetii is
small, three rare codons in the chromosome and none in the plasmids are less used
than 10%. As expected the A+T rich plasmids (60.33-60.70%) revealed a remarkable
bias toward U- and A-ending codons. GC3s values range from 0.23 to 0.59 (mean
0.351) for the plasmids and from 0.138 to 0.506 (mean 0.399) for chromosomal genes
with an average A+T content of 56%. Nc values range from 40.2 to 61 (mean 53.43)
for plasmid genes and 39.81 to 58.39 (mean 51.29) for chromosomal genes. Thus,
SCU bias for C. burnetii genes is generally low and is possibly correlated with a low
gene expression level.
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71A
A hypothetical nature of the obligate rickettsial parasitism.
Victor V. Emelyanov.
Gamaleya IEM, 123098 Moscow, Russia
90
Rickettsia prowazekii, causative agent of epidemic typhus, is one of a few obligately
intracytoplasmic bacteria and, thus, shares a common niche with mitochondria. Evolutionary
studies based on several molecular chronometers show that mitochondria share last common
ancestor with rickettsiae. A groE operon of R. prowazekii was recently sequenced (see
accompanying paper), and inferred Hsp10 and Hsp60 heat-shock protein sequences were first
used in phylogenetic analysis (PHYLIP 3.5c package). Hsp60 data set (51 spp.) included as
many as possible members of the family Rickettsiaceae and mitochondrial-type chaperonins
from Protozoa. It was shown by bootstrap analysis (FITCH consensus tree) that rickettsial
homologue occurs 95% of the time as the least and the earliest diverging member within the
cluster of Rickettsiaceae - a sister group of the mitochondrial clade. The genus Rickettsia was
followed by Orientia, next by Ehrlichieae/Wolbachieae. Of interest, Holospora obtusa, obligate
endosymbiont of protist, diverged (98%) well before the mitochondria/obligate intracellular
bacteria common lineage. Hsp60-based analysis is very robust since results depend on neither
alignment mode nor phylogenetic program used. Being somewhat unstable, Hsp10 tree
topology generally agrees with one of Hsp60 while shows even affiliation of H. obtusa to the
prior diverging cluster of epicellular and free-living alpha-Proteobacteria. Like Hsp60, SSU
rRNA analysis performed by DNAML program showed divergence of protist’s parasite prior to
mitochondrion/Rickettsiaceae. Here I present an idea based essentially on evolutionary
considerations that easily explains inability of rickettsiae to replicate on bacteriological media
and slow growth within eukaryotic cells. It is suggested that like mitochondria the rickettsiae
import some required proteins from the cytosol what may be a molecular basis of the obligate
intracellular parasitism. One can suppose that about 2 billion years ago some rickettsia-like
microorganism had become an endosymbiont of a progenitor of eukaryotic cell, and perhaps
had lost some genes. This bacterium has given rise to both rickettsiae and mitochondria. While
the organelles progressively lacked the genes and concomitantly developed fine protein import
machinery, the rickettsiae continued to be visitors, and had lost maybe a few genes the
products of which they could import from eukaryotic cell.
A Coxiella burnetii penicillin-binding protein
Anna Macellaro, Karin Hjalmarsson, and Lena Norlander*
Division of Microbiology, Department of NBC Defence, Defence Research
Establishment, S-901 82 Umeå, Sweden
Penicillin-binding proteins (PBPs) are cell wall located enzymes falling into two
distinguishable groups, the low and the high molecular mass PBPs. The low
molecular mass PBPs are monofunctional enzymes involved in the remodelling of
peptidoglycan during cell growth, while the high molecular mass PBPs are
multimodular enzymes and the main enzymes responsible for cell wall peptidoglycan
assembly.
A Coxiella burnetii homologue of high molecular mass PBPs has been
identified. These PBPs are essentially composed of two modules, the penicillinbinding and the non-penicillin-binding modules, which are linked to each other in a
single polypeptide chain that folds on the outer face of the plasma membrane and is
anchored into the membrane by an uncleaved amino-terminal signal peptide. The two
modules of PBP are probably in close interaction and form interdependent folding
entities. Depending on the motifs present in the non-penicillin-binding modules, PBPs
fall into classes A and B.
Based on amino acid sequence comparisons the Coxiella burnetii PBP belongs
to the class B PBPs, i. e. subclass B3 represented by Escherichia coli PBP3. The
Coxiella burnetii PBP comprises the four motifs (motifs 1 to 4) characteristic of the Nterminal non-penicillin binding module. Furtherrnore, the amino acid sequence
signatures of the modules are found in the Coxiella burnetii sequence. Two of these
signatures, in the motif 1 and 3, are key elements of the amino acid sequence-folding
information for E. coli PBP3.
- 13 -
91
Molecular typing of granulocytic Ehrlichia species by intergenic
spacer sequence analysis.
104
Dionysios Liveris*, Ira Schwartz, Gary Wormser, Maria Aguero-Rosenfeld and
Thomas Daniels
New York Medical College, Valhalla, NY 10595.
Genetic diversity of the tick-borne agent of human granulocytic ehrlichiosis
(HGE) was assessed in HGE patient specimens and Ixodes scapularis ticks from
Westchester County, New York. The 16S-23S rDNA intergenic spacer and a region of
the groES-groEL operon, including the intergenic spacer, were amplified by PCR and
sequenced. Among 15 HGE clinical isolates, 9 of which were cultured in HL-60 cells
and 6 of which were amplified directly from blood, no sequence differences were
observed at either genetic locus. Identical sequences were also observed for two
human isolates from Connecticut, one from Long Island, NY and E. equi. In contrast,
rDNA spacer analysis of directly amplified sequences from 10 ticks revealed
sequence heterogeneity ranging from 0-3.3%. Surprisingly, for these same tick
specimens, the sequenced portions of groES-groEL were completely conserved. This
striking lack of sequence polymorphism among human HGE isolates suggests a
relatively recent introduction of this agent into the pool of human pathogens. The fact
that only a subset of HGE isolate spacer sequence types observed in ticks are found
in patients raises the intriguing possibility that not all isolates of HGE in ticks may be
pathogenic for humans.
Genomic subtraction of two Bartonella henselae strains
Christian Ehrenborg and Martin Holmberg
Section for Infectious Diseases, Department of Medical Sciences, Uppsala University
Hospital, Uppsala, Sweden.
Previous characterisation of B. henselae strains have shown heterogeneity in
gene sequences and fingerprinting patterns. Phenotypic variation has also been
observed between some isolates. We have studied two strains with different antigenic
properties, and gene sequence differences - the Houston-I (ATCC 49882) and
Marseille (URLLY 8) isolates.
The two genomes were subtracted against each other, using both of them
either as driver or tester DNA. The subtractions were performed using PCR-Select
bacterial genome subtraction kit (Clonetech, Palo Alto, Calif. U.S.A.), and a
subtraction library was constructed. Several clones were selected, and differentially
hybridized against the two genomes.
Two clones with DNA fragments only present in the Marseille strain have been
found in a first screening, and are being further characterized.
We show here that the Marseille strain contains genetic material not present in
the type strain Houston I. Characterization of clones from the subtraction library will
shed light on the nature of this genetic material, and additional B. henselae strains are
being tested for presence of these sequences.
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116
Analysis of the interventing sequences in 23s rRNA genes of the
chinese Coxiella burnetii isolates
133
Rong Chen, Bohai Wen*, Shurong Yu
Department of Microbiology, The Third Military Medical University, Chongqing
400038, P. R. China
The intervening sequences (IVS) carried by 23S rRNA genes of Coxiella
burnetii (Cb) were amplified from seven Chinese isolates and two foreign reference
strains by polymerase chain reaction (PCR) and the amplimers were directly
sequenced by dideoxynucleotide methods. The IVSs of the isolates from different
locations and sources in this study, except three isolates from Yunnan province, were
identical. The IVSs of three Yunnan isolates from human and sheep were identical
and they were one 7-base repeat unit less than that of the other 6 isolates. Our results
demonstrated that the IVS of Cb is a highly conservative element in chromosome and
it is different from its homologous elements existing in the chromosome of Spirochete
which are highly divergent among strains; The difference of Cb IVSs is related to the
geographic distribution of the organisms. A nested PCR was established based on the
Cb IVS, and it was demonstrated to be highly specific and sensitive to identify Cb in
different speciments.
Evaluation of Coxiella burnetii lipopolysaccharide components as
the immunodiagnostic reagents.
Hussein A, Kováèová E, Ftáèek P, Toman R.
Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic
Lipopolysaccharide (LPS) has been considered to be a major factor of virulence
expression and infection of Coxiella burnetii. It is a predominant surface antigen
capable of inducing antibodies. Biologically active epitopes in the LPS are potential
candidates for immunodiagnostic purposes. Various fractions of the LPS and the Opolysaccharide chain were isolated by combination ofgel-chromatography and HPLC.
The purified fractions were analyzed for chemical composition by both colorimetric
methods and GC-MS, and their molecular heterogeneity was investigated by SDSPAGE and TLC. The utility of the purified fractions in ELISA was evaluated by testing
their activities with animals and human sera and comparing these reactivities with that
of native LPSs. The results indicate that ELISA with some of these fractions is a
sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.
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140
Transformation of Coxiella burnetii to quinolone-resistant strain.
detection of a new pefloxacin resistance mutation in Coxiella
burnetii gyrA by direct DNA sequencing and PCR-RFL.
144B
Ioanna Spyridaki, Anna Psaroulaki, Ana Aransay, Efstathia Scoulica, Yannis
Tselentis.
Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical
Medicine, Faculty of Medicine, University of Crete, Heraklion, Greece.
To characterize mechanisms of resistance to fluoroquinolones by Coxiella burnetii, a
total of 12 strains (8 Greek isolates from acute Q fever patients, two reference strains Nine Mile,
Q212 and two pefloxacin resistant laboratory strains) were examined for the presence of point
mutations in the quinolone resistance determining region (QRDR) of gyrA gene. Their QRDR of
gyrA gene was amplified by the polymerase chain reaction (PCR) assay and sequenced to
identify mutation in gyrase A. The PCR assay, followed by restriction fragment length
polymorphism (RFLP) analysis, confirmed the results obtained by direct DNA sequencing and
detected the gyrA mutation. The gene sequences of all of the 8 Greek isolates and the two
reference strains Nine Mile and Q212 were identical. One type of mutant was obtained in gyrA
in both pefloxacin resisted laboratory strains. This single gyrA mutation caused a much greater
increase in the MICs of fluorokinolones. The increase in the minimal inhibitory concentration
(MIC) was 32-to 64-fold in ciprofloxacin and pefloxacin of resistant laboratory strains, compared
with the MICs of sensitive Greek isolates and reference strains. The two pefloxacin resistant
laboratory strains with high-level MICs of ciprofloxacin and pefloxacin showed a point mutation
at codon 87. The nucleotide mutation (G→A) lead to the substitution of glutamic acid (codon
GAG) by lysine (codon AAG ) at a position corresponding to aminoacid 87 of E. coli. These
results indicate that the high-level resistance to ciprofloxacin and pefloxacin of pefloxacin
resistant laboratory strains is associated with a nucleotide mutation in gyrA. The PCR-RFLP
proved to be a simple, rapid, and useful method for detecting the gyrA mutation associated with
pefloxacin resistance in C. burnetii.
Characterizing the genetics of a bacteriophage-like particle from
Bartonella bacilliformis.
KD. Barbian and M.F. Minnick
The University of Montana, Missoula, MI.
Bartonella bacilliformis and B. henselae, agents of Oroya fever and cat-scratch
disease, respectively, produce bacteriophage-like particles (BLPs) that package 14kbp segments of the host chromosome. RFLP and southern blot analysis of the BLP
DNA from B. bacilliformis suggest that packaging is much less random than observed
in B. henselae phage. Data also suggest that the linear, double-stranded, BLP DNA
molecules have non-covalently closed ends with 3' overhangs. The 3' overhangs are
not complementary to one another, as ligation attempts have been unsuccessful.
Additionally, BLP DNA molecules appear to be packaged immediately upon synthesis
as there is no unpackaged phage DNA present within the cell. To determine if BLPs
contribute to horizontal gene transfer, mutants of B. bacilliformis were generated by
allelic exchange with a suicide vector construct termed pKB1. pKB1 contains a pMB1
origin of replication, a kanamycin resistance cassette (nptI), and an internal fragment
of the 16S-23SrDNA intergenic spacer region. Homologous recombination between
pKB1 and one of the three rRNA operons present in the B. bacilliformis chromosome
led to its disruption and produced a kanamycin-resistant phenotype. Southern blot
analysis confirmed that allelic exchange had occurred. Furthermore, it was known that
BLPs from some of these strains were able to package the mutagenized region
containing the kanamycin resistance cassette. However, numerous attempts at
transduction with BLPs from these strains were unsuccessful. We are currently
analyzing the phage genome in an effort to discover the origin of replication for the
BLP DNA in hopes of harnessing the phage as a genetic tool.
- 16 -
146B
Genetic comparison among naturally occurring Ehrlichia equi and
Human Granulocytic Ehrlichiosis (HGE) agent isolates from
northern California based on the nucleotide sequences of 16S
rRNA, 444 Ep-ank Repeat and Heat Shock Protein (gro-ESL) Genes
149
Joon-seok Chae, Janet Foley, J. Stephen Dumler*, John E. Madigan.
Department of Medicine and Epidemiology, School of Veterinary Medicine, University
of California, Davis, California 95616. *Department of Pathology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
We have examined the sequences of three genes to determine the genetic diversity among strains
from 11 naturally occurring clinical cases of Ehrlichia equi in horses and 2 HGE agent infections in humans
in the far west. Ehrlichia equi isolates were obtained from Sierra (n=6), Mendocino (n=3), Sonoma (n=1)
and Marin (n=1) counties and Human Granulocytic Ehrlichiosis (HGE) agent isolates were obtained from
Humboldt county (n=2) in California. These isolates were analyzed by determining the sequences following
polymerase chain reaction using specific primers for 16S rRNA, heat shock protein (gro-ESL) and 444 Epank repeat genes. All three genes were amplified from 13 clinical samples. The nucleotide sequences of
16S rRNA genes were identical among two humans isolates (CAHU-HGE1 and CAHU-HGE2) from
Humboldt county and the sequences of two horse E. equi isolates (CAMEBS and CAMAWI) from
Mendocino and Marin counties. But, there was a single nucleotide difference at positions 180 and 34
between the E. equi isolates from Sonoma (CASOLJ) and Sierra (CASITL) counties, respectively, and the
other E. equi isolates. The sequences of 444 Ep-ank repeat gene fragment which included the region
encoding the four ankyrin repeats were identical among all E. equi and two HGE agent isolates. These
sequences when compared to the same gene sequences of a HGE agent isolate from the eastern United
States (BDS strain) had two differences at nucleotide positions in 326 and 398. When the heat shock
protein (gro-ESL) gene sequences were compared, one CAHU-HGE1 and three E. equi isolates (CASITL,
CAMEBS; Mendocino and CAMAWI; Marin county) had identical sequences and the other isolates had
differences in two and four nucleotide positions, 1,483 and 1,546 between CAHU-HGE1 and CAHU-HGE2
isolates and 347, 1,303, 1,448 and 1,496 between CAHU-HGE1 and CASOLJ isolates, respectively. These
results suggested that the horse E. equi (CAMEBS and CAMAWI isolates) and human HGE agent (CAHUHGE1 isolate) from northern California were identical species based on 16S rRNA gene and 444 Ep-ank
repeat and gro-ESL gene nucleotide sequences.
Study on target gene of new type Rickettsia tsutsugamushi in
China by pcr/ rflp and sequence analysis.
Guo Hengbin, et al .
Institute of Military Medicine,Nanjing Command,PLA,Nanjing 210002
In order to clarify the characteristic of the target gene sequence ofthe epidemic
strain of Rickettsia tsutsugamushi (Rt) in Jiangsu province,the group and type pirmers
were constructid from Rt type-specific antigen 56kDa(Rt-tsa56kDa) gene of this
prototype strains Rt, PCR was used for detecting and typing of Rt; the amplification
produsts by group primers by RFLPand sequenced.The results showed that these
primer possessed the characteristic of Rt; the epidemic strain Rt in Jiangsu province
was similar to kawasaki strain of Japan;there were no Hha Isite in the target gene of
Rt ; the homolgy of the sequence of Rt strain in Jiangsu province was 96.87% to
kawasaki strain, but it was 52.00% to other prototype strains;the Hha I site was
mutated from GCGC to GTGC.The results indecati that the Rt strains in Jiangsu
province belong to the kawasaki strain of Japan, but there is hereditary variation.so it
is a new type Rt probably in China.
- 17 -
165A
The htrA gene of Bartonella henselae restores E. coli htrA mutant
B178 the ability to grow at 42°C.
169
Sandra I. Resto-Ruiz* and Burt E. Anderson
University of South Florida, College of Medicine, Department of Medical Microbiology
and Immunology, Tampa, FL.
The high temperature requirement A (HtrA) protein is among the bacterial
stress response families responsible for the regulation of the extracytoplasmic heat
shock response. Bartonella henselae (Bh) htrA bears homology with the htrA genes of
E. coli (Ec) and B. abortus (Ba). Also, homologous PDZ domains, which presumably
mediate protein target recognition, were found in the htrA proteins of Ec, Ba, and Bh.
However, the putative hsp function of Bh HtrA has not been demonstrated. To assess
the function of the Bh htrA gene, we performed genetic complementation studies
using the Ec htrA mutant B178:mini Tet whose growth is impaired at 42°C.
Electrocompetent B 178 cells were transformed with constructs derived from clones 1,
4, and 7 harboring pUC19/BhhtrA (1= whole BhhtrA), pUC19/BhhtrA (4=BhhfrA
excluding promoter area), and pUC19/BhhtrA:kanR (7=BhhtrA kanamycin mutant),
respectively. The ability to grow at 42°C was restored in cells transformed with BhhtrA
clones 1 and 4. Growth at 42°C was still impaired in cells transformed with either
pUC19 only (negative control) or the Bh htrA kanamycin mutant as compared to
parallel cultures at 30°C. These results suggest that the product of BhhtrA functions as
a stress response protein which enables cell survival upon heat shock.
Conservation of the 17-kDa antigen throughout the genus
Bartonella.
Debra Sweger*, Sandra Resto-Ruiz, Noel Hawke and Burt E. Anderson.
University of South Florida, College of Medicine, Department of Medical Microbiology
and Immunology, Tampa, FL U.S.A.
Bartonella henselae encodes a 17-kDa antigen that has been shown to elicit a
strong immune response in patients with Cat Scratch Disease (CSD). A fusion
construct of this gene has been useful in screening human serum samples for CSD. In
this study, PCR assays were performed on several species of Bartonella with
oligonucleotides prepared from Bartonella henselae (Houston-1) sequence data to
determine if they harbored a homolog of the 17-kDa antigen gene. Amplicons of
varied sizes were demonstrated when using the following chromosomal DNA
templates: B. henselae, B. quintana, B. elizabethae, B. bacilliformis, B. vinsonii and
B.berkhoffi. Further investigation of these amplicons included DNA sequencing,
ligation of the amplified products into pUC19 and subcloning for protein expression of
the 17-kDa gene homolog. Immunoblot analysis and further testing should reveal the
potential role of these homologs as diagnostics reagents for related Bartonella
diseases.
- 18 -
170
Characterization of the Bartonella henselae gene encoding the 43kda outer membrane endothelial cell binding protein.
171
Andrew W. O. Burgess and Burt E. Anderson.
University of South Florida College of Medicine, Dept. of Medical Microbiology and
Immunology, Tampa, Florida 33612.
Members of the genus Bartonella are unique in that they are bacteria that cause
proliferation of microvascular endothelial cells and neovascularization (angiogenesis).
The mechanisms by which Bartonella henselae causes these processes are
unknown. Previous work in our laboratory showed that the 43-kDa outer membrane
protein (Omp43) is the major adhesin for endothelial cells. The gene encoding Omp43
was cloned and sequenced. Sequence analysis revealed an open reading frame of
1206 nucleotides coding for a protein of 402 amino acids. Analysis of the deduced
amino acid sequence shows 38% identity over the entire sequence to a Brucella spp.
porin, as well as shows a potential signal sequence and peptidase cleavage site,
further supporting the outer membrane location of Omp43. Cleavage of the signal
peptide would result in a mature 380-aa polypeptide with a predicted MW of 42 kDa.
Western blot analysis of the expression of 0mp43, without the signal sequence, in
Escherichia cou revealed a histidine-tagged fusion protein of 45 kDa which retained
the ability to bind endothelial cells. Current research is aimed at expressing Omp43 as
four smaller peptides ranging from 15-18 kDa for further analysis of the binding
properties of Omp43. The cell specificity of Omp43 is also under investigation, as well
as identifying the endothelial cell receptor for Omp43. The cloning of Omp43 should
provide a tool for further investigation of the role of adherence in the pathogenesis of
B. henselae.
Cloning of Orientia tsutsugamushi 47kda antigen gene.
Qiang Yu, Chen Xiangrui, Zhang Yongguo, Niu Hua, Zhang Xueyi
Institutes of Microbiology and Epidemiology , Beijing ,100071
Orientia Tsutsugamushi have 150,110,72,58,56,47, and 22kDa antigen gene,
the 56 and 58kDa antigen gene is the most abundant of antigen content, it is major
protein antigen. Moreover, its immunology protection is not satisfaction. Some author
find that there is stronger protecting immune of Orientia Tsutsugamushi 47kDa
antigen,he think that 47kDa antigen of Orientia Tsutsugamushi can be used vaccine
candidate. According to the ideal, we design pair primer depends on template of
47kDa-antigen gene sequence from EMBL and amplified 47kDa-gene segment. The
production of PCR ligase to T-vector, and then subclone to PBV220 vector, we get
125 recombinant. Three recombinant have specific gene of Orientia Tsutsugamushi
identified by PCR, EcoRI and BamHI. The recombinant express by heat shock, the
specific protein is seen by SDS-PAGE electrophoresis.
- 19 -
172A
Coxiella burnetii induce the reorganization of actin cytoskeleton in
human monocytes
S. Meconi*, V. Jacomo*, P. Boquet§, D. Raoult*, J. L. Mege*, C. Capo*
* Unité des Rickettsies, Faculté de Médecine, Marseille, France - § INSERM Unité
452, Faculté de Médecine, Nice, FRANCE
Virulent Coxiella burnetii, an obligate intracellular bacterium is poorly
internalized by monocytes as compared to avirulent variants. A differential
mobilization of actin cytoskeleton may account for this distinct phagocytic behavior.
Scanning electron microscopy showed that virulent C. burnetii stimulated profound
changes in morphology of THP-1 monocytes consisting of membrane protrusions and
polarized projections. These changes were transient, requiring 5 min to reach a
maximum value and vanishing after 60 min. Avirulent variants of C. burnetii did not
induce any significant morphological change. The distribution of filamentous actin (Factin) was then studied, using a specific probe, bodipy phallacidin. Virulent C. burnetii
induced a profound and transient reorganization of F-actin, accompanied by an
increase in the F-actin content of monocytes. F-actin was colocalized with myosin in
cell protrusions, suggesting that actin polymerization and the tension of actin-myosin
filaments play a role in C. burnetii-induced morphological changes. Using the C3
exotransferase of Clostridium botulinum, which selectively inhibits the activity of the
GTPase Rho, we showed that the Rho protein probably regulates the morphological
changes and the reorganization of F-actin stimulated by C. burnetii. In addition, the
cell-bacterium contact seems necessary to induce cytoskeleton reorganization.
Virulent bacteria were found in close apposition with F-actin protrusions. The
manipulation of the actin cytoskeleton by C. burnetii may play a critical role in their
internalization strategy.
- 20 -
197A
Monday June 14th
Slide session 1
Bacteriology and Genetics
A tail-specific protease from Bartonella quintana may be involved
in hemin acquisition
M.F. Minnick1, L.S. Smitherman,1 & J.A. Carroll2
The University of Montana, Missoula, MT1, & Rocky Mountain Labs, NIH, Harnilton,
MT2
Early work by Myers, Cutler and Wisseman showed that B. quintana has the
greatest in vitro hemin requirement for any known bacterium, at 20-40 µg/ml of
medium. With this characteristic in mind, we investigated the molecular basis for
hemin acquisition by the pathogen. Cell preparations of B. quintana (Oklahoma 90268) exhibited a pronounced brown color, indicating that hemin was accumulated by
the bacterium as it grew on blood agar plates. Hemin uptake was markedly
pronounced, even when compared to three other pathogenic Bartonella spp. A
genomic library of B. quintana was prepared in pACYC 177 using E. coli DH5 as the
host. Screening for hemin-binding colonies produced two sibling strains containing the
same plasmid, termed pHB2. The pHB2 insert is 4474 bp in length and contains
ORF's that are homologous to a virulence gene cluster from B. bacilliformis, encoding
the filament-A protein (fiIA), tail-specific protease (ctpA), and the invasionassociated
locus (ialA and ialB) proteins. The hemin-acquisition phenotype was mapped to the
protease gene using linker mutagenesis. In addition to hemin uptake, the B. quintana
ctpA also conferred a Congo red-binding phenotype upon E. coli, but did not affect
uptake of phenol red or Sudan black, another lipophilic dye. Hemin uptake was
significantiy enhanced vhen ctpA was expressed from a single copy-number vector,
suggesting that the phenotype is biologically relevant to B. quintana. Hemin blot
analysis indicated that the protease does not bind hemin directly, but may instead
alter the cell's permeability to hemin and hemin-like molecules, such as Congo red. E.
coli expressing ctpA display a fliamentous morphoiogy, reminiscent of penicillinbinding protein 3 mutants. [ctpA- GenBank accession # AF 110497].
- 21 -
146A
Molecular analysis of strain-specific protective antigen and
GROEL homologue protein of Ehrlichia risticii
10
S. K. Dutta, B. Biswas and R. Vemulapalli
Virginia-Maryland Regional College of Veterinary Medicine - University of Maryland,
College Park, Maryland 20742, USA
Ehrlichia risticii causes Potomac horse fever (PHF), an important disease of equine,
recognized in the United States, Canada, and other countries in the world. The disease shows
severe clinical signs, and the mortality can reach as high as 10-20%. Vaccination with activated
vaccines is performed regularly, particularly in endemic areas. In spite of this, high numbers of
vaccine failure are a constant occurrence, which has been clearly established. Among other
causes, the emergence of variant strains, which has been well-documented, has been
associated with the vaccine failure. Molecular analysis of two strains of E. risticii, the 25D strain
isolated in 1984 during the initial recognition of PHF and the 90-12 strain isolated in 1990 from
a vaccinated horse suffering from clinical PHF, indicates the presence of a immunoprotective
strain-specific antigen (SSA) of 50kD and 85kD respectively. These two SSAs are homologues
of their respective strains as demonstrated by their sequence analysis and serological cross
reactivity. The genes of these SSAs contain varying number of tendem repeats that contribute
to a constant-number of common amino acid domains (AADs) and varying number of unique
AADs in their respective SSAs. However, the arrangement of the common AADs in these two
SSAs is totally different. The recombinant 50kD SSA provided homologous protection only,
whereas the 85kD SSA provided both homologous and heterologous protection in mice. The
SSA of field strains varies from 48kD to 85kD. The component 55kD antigen is present in a heat
shock operon along with the gene for a ~10kD protein which are homologues of E. coli GroEL
and GroES proteins respectively. In contrast to SSA genes, there was no nucleotide sequence
difference between the genes of the 55kD antigen, nor the entire operons from both E. risticii
strains.
Evaluation of protective capacity of Cowdria ruminantium genes
encoding immunogenic proteins by Dna immunization
S.M. Mahan1,2, A. Nyika1, B. Simbi1, A. Moreland2, M. Bowie2, A. Lundgren2, H.
Kolenda-Roberts2, L.M. Lew2, C. McCranie2, J. Palmer2, T. McGuire3, F.
Rurangirwa3, M. J Burridge2 and A. F. Barbet2
1 UF/USAID/SADC Heartwater Research Project, Zimbabwe; 2 University of Florida,
Gainesville, Florida; 3 Washington State University, Pullman.
Ten cloned genes encoding immunogenic proteins of C. ruminantium were
evaluated for protection against lethal C. ruminantium challenge in DBA/2 mice by
DNA immunization. The MAP 1 gene of C. ruminantium protected mice against
homologous challenge, by inducing Th1 type cellular responses characterized by
-2. These responses were enhanced by boosting DNA
vaccinated mice with homologous recombinant protein which augmented protection
against challenge. An additional 9 genes of C. ruminantium were similarly tested.
Preliminary data suggest that another 5 genes can confer protection against lethal
challenge in terms of prolongation of life. The full protective capacity of these genes
will become apparent after studies which will include boosting with each respective
antigen. Implications of these findings for development of recombinant vaccines
against C. ruminantium infection will be discussed.
- 22 -
12A
Differential expression of transcriptional
elements by Coxiella burnetii life cycle stages
and
translational
65
R.Seshadri, L.R. Hendrix, J.E. Samuel.
Texas A&M Health Science Center.
Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular organism that resides in an acidified
phagolysosome and has remarkable ability to persist in the extracellular environment. A proposed mechanism allowing
survival in both extra- and intracellular environment suggests C. burnetii has evolved a developmental cycle that includes
at least two morphological forms designated large celI variants (LCV) and small celI variants (SCV) that have distinct
ultrastructural and antigenic characteristics. We hypothesize that these differences result from differential gene expression
and that identification and characterization of these genes will allow prediction of the functional roles of life cycle stages. To
characterize differentially expressed genes, we adopted a two-fold strategy: First, a panel of monoclonal antibodies,(Mab)
were compared by Western blot for rcactivity with LCV and SCV antigens and a 35 kDa reactive (LCV up-regulated/Mab
NM7.3) and 45 kDa reactive(LCV-specificIMab NMl 83) antigen were identified. NM7.3 was used to screen a Zap II. C.
burneiji DNA expression library and an immunoreactive clone was identified with sequence similarity to an rpsB-tsf gene
cluster that encodes a ribosomal protein S1 and elongation factor EF-Ts. A similar screen with NM183 did not identify
immunoreactive clones. An alternate strategy to clone this antigen was devised based on the following observations: 1)
NM183 strongly crossreacted with a 45 kDa antigen from Chlamydia trachomatis, and 2) NM183 reacted with purified
His4agged Chlamydial EF-Tu suggesting that the LCV-specific immunoreactive antigen was EF-Tu from C. burnetii. The
highly conserved nature of EF-Tu among bacteria allowed the design of degenerate PCR primers to amplify a 900 bp
internal region of a putative tuf gene from C. burnetii. Data indicated that C. burnetii have tufA and tufB homologues. Based
upon the organization of tuf genes from other bacteria, upstream primers were designed which amplified most of the tufB
gene, including its Nterminal region. This open reading frame was shown to express a NM183-reactive product confirming
that the antigen was EF-Tu. Because of the LCV-specific up-regulation of translational factors and previous data identifying
two histone-like proteins specific for SCV, we hypothesized that LCV and SCV are comparable to log phase and stationary
phase growth stages. Since sigma factors control this transition, we speculated that a rpoS homologue would control SCV
gene expoession. To test this, the second part of this study was designed to clone and compare sigma factor expression in
C. burnetii. Colony hybridization using a PCR amplified internai rpoD-like region identified the majority of the rpoD gene
from a plasmid library of C. burnetu DNA. PCR amplification did not yield rpoS-like product, but Legionella pneumophila 's
rpoS gene did hybridize with a 4kb ilindili fi~rnent of chromosomal digest confirming the ability of this probe to recognize
clones which may contain a rpoS homologue from C. burnetii. By proving differential expression of key elements of the
transcriptional and translational machinery, we hope to better understand the function of the LCV and SCV and to develop
a more accurate life cycle model for C. burnetu.
Cloning and characterization of a Heat Shock sigma32 Homolog in
Coxiella burnetii.
J. Seshu*, Paul L. Dexter and Louis P. Mallavia
Department of Microbiology, Washington State University, Pullman, WA 99164-4233,
USA
Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular
pathogen that survives and replicates in the phagolysosomes of macrophages. Heat
shock proteins are a class of highly conserved proteins that enable organisms to cope
with a variety of environmental and physiological stresses. Since the transcription of
heat shock genes is under the control of sigma 32 (rpoH gene product), the purpose
of the present study is to identify and characterize the rpoH gene of C. burnetii.
Screening of several C. burnetii genomic DNA libraries resulted in the identification of
a recombinant plasmid designated pJS103 that contained an open reading frame
capable of encoding a protein of 288 amino acids with a calculated molecular mass of
31.6 kDa. The deduced amino acid sequence of this open reading frame shared
similarity with that of RpoH, the transcriptional regulator of several heat shock
proteins, from a number of bacteria. An E. coli rpoH- mutant KY1608 was successfully
complemented with pJS103 indicating that the C. burnetii gene encoding the 32 kDa
protein is a functional homolog of the rpoH gene from E. coli. Polyclonal antisera
-Galactosidase-CbRpOH fusion protein showed reactvity to a 32 kDA protein
of C. burnetii Nine Mile phase I by Western blot analysis. Exposure of C. burnetii to
42oC in a low pH axenic medium resulted in the upregulation of RpoH as revealed by
Western blot analysis using anti-CbRpoH sera. Identification and characterization of
the rpoH gene of C. burnetii will help to elucidate the regulation of different genes
which are under the transcriptional control of sigma 32 factor.
- 23 -
145
Monday June 14th
Poster session 2
Physiopathology and Immunity
Characterization of the Major Outer Membrane Protein of Coxiella
burnetii
Sunita E. Varghees, Katalin A. Kiss, James E. Samuel
Department of Medical Microbiology and Immunology, Texas A&M University Health
Science Center
Coxiella burnetii, the etiologic agent of Q fever, appears to have three primary
morphologic variants designated large cell variant (LCV), small cell variant (SCV), and
small dense cell (SDC). Earlier preliminary studies suggested that P1, the 29.5 kDa
major outer membrane protein (MOMP) of the organism, may function as a porin and
may be differentially expressed by these variants: highly expressed in LCV,
downregulated in SCV, and not apparent in SDC. Outer membrane surface
localization studies were performed to confirm monoclonal antibodies (Mabs), which
recognized a 29 kDa C. burnetii protein, were P1-specific using immunoprecipitation
of 125I-labeled surface proteins and immunofluorescence (IFA). The differential
expression pattern previously observed was then evaluated using IFA, immunogold
electron microscopy and Western blotting. Next, a protocol was developed to purify
the MOMP using the zwitterionic detergent Empigen. One and two-dimensional gel
electorphoresis was used to confirm the relative homogeneity of the resulting P1
protein. N-terminal analysis of P1 generated an amino acid sequence for cloning
studies. Finally, the porin activity of purified P1 is being evaluated using a planar black
lipid bilayer assay. Confirmation of both porin activity and differential expression of P1
will allow for a better understanding of potential life cycle roles of the morphologic
variants of C. burnetii.
- 24 -
30
A comparative study of the actin-based motilities of the
pathogenic bacteria Listeria monocytogenes, Shigella flexneri, and
Rickettsia conorii.
36
E. Gouin1, H. Gantelet1, C. Egile2, I. Lasa1*, H. Ohayon3, V. Villiers1, P. Gounon3,
P.J. Sansonetti2 and P. Cossart1
1Unité des Interactions Bactéries-Cellules, 2Unité de Pathogénie Microbienne
Moléculaire, 3Station Centrale de Microscopie Electronique, Institut Pasteur, 25 and
28 Rue du Dr Roux, 75724 Paris Cedex 15, France - Present address: Dpto.
Producción agraria, Universidad Pública de Navarra, Campus de Arrosadia s/n,
Pamplona-31006, Spain.
Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial
pathogens that are able to polymerize actin into 'comet tail' structures and move within the
cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are
known to require the bacterial proteins ActA and IcsA, respectively and several mammalian
cytoskeleton proteins including the Arp2/3 complex and VASP (Vasodilator-stimulated
phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich
syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In
the present study, we have analysed the actin-based motility of this bacterium in comparison to
those of L. monocytogenes and S. flexneri. Rickettsia moved at least three times slower than
Listeria and Shigella in both infected cells and Xenopus Iaevis egg extracts. Decoration of actin
with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia
have a structure strikingly different from those of L. monocytogenes or S. flexneri, with longer
actin filaments devoid of any branching. Immunofluorescence studies revealed that the two host
proteins, VASP and a-actinin colocalized with actin in the tails of Rîckettsia but neither the
Arp2l3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in
Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different
mechanism of actin polymerization.
Characterization of a virulence gene homologue in Bartonella
henselae
Michael Schmiederer and Burt E. Anderson.
University of South Florida, College of Medicine, Department of Medical Microbiology
and Immunology.
Bacterial virulence genes are commonly found in clusters referred to as
pathogenicity islands, and in Bartonella henselae a similar arrangement may be
observed. In 1995 Anderson, et al described a 17kDa antigen of B. henselae Hou-1.
The clone H13/SOLR that contained this gene also contained a partial open reading
frame designated ORF1, which is 459 nucleotides long and is directly upstream of the
17kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 to
other know genes in Genbank revealed significant homology with several other
bacterial virulence genes, including VirB4 of Agrobacterium tumefaciens virB operon
(56/149 amino acids), PtlC (36/84 amino acids) of the Bordetella pertussis toxic
liberation (ptl) operon, and PicB of Helicobacter pylori cagA operon (33/78 amino
acids). The H. pylori picB gene, which is part of the cagA pathogenicity island, is 2655
nucleotides in length and codes for a 101kDa protein. An overlapping clone,
pGB3/XLOLR was recovered by immunoscreening of a B. henselae Hou-1-ZapII
expression library. Sequence analysis and restriction digestion established that pGB3
contains the 17kDa gene and a 3kb region upstream. In vitro transcription and
translation data demonstrated that pGB3 coded for a protein of 90kDa. The position of
this 3kb region on pGB3 in relation to the 17kDa gene and the similarity in size
between PicB and the pGB3 protein suggests that a significant part of, if not the
entire, ORF1 is contained at the 5’end of pGB3. Sequencing of this region is currently
underway to determine the entire sequence of the open reading frame and the
deduced amino acid sequence.
- 25 -
39
Serologic analysis of immune response in humans vaccinated
against epidemic typhus and subsequent response to Rickettsia
prowazekii.
50
Ignatovich V, Eremeeva M, Kekcheeva N, Genig V, Penkina G, Umnova N,
Tupicin A, Belousova L.
Gamaleya Research Institute, Moscow, Russia.
These studies summarize the results of 40-year serosurvey of anti-R.prowazekii
antibodies (Abs) in sera of laboratory employees, who had been vaccinated against
epidemic typhus (ET). Immunization was performed with different vaccines and
schedule. Sera were collected at I,6,I2 months and then annually. Complement
fixation (CF), microimmunofluorescence (MIF), indirect hemagglutination test with
protein (IHA-P) and lipopolysaccharide (IHA-LPS) antigens were used for serum
analysis. A correlation between the schedule of vaccination, contacts with viable
R.prowazekii and occurence of laboratory acquired ET has been established.
Serological analysis in vaccinated and laboratory acquired vaccinated people
revealed considerable differences in dynamics of specific Abs. It mainly concerns
CFA. In the vaccination group CFA could be detected for not more than 1,0-2,0 years.
In vaccinated and exposed individuals CFA persisted up to 40 years. MIF and lHa-P
Abs decreased quikly and gradually in both groups respectivily. Serosurvey
 revealed that out of 48 laboratory acquired vaccinated people in 10 CF Abs
persisted in titres 1:I0 - 1:40 for 8-28 years after exposure and than disappeared.
However MIF and IHA-P Abs could be detected for 5-I0 years more.These results
suggest that there is a potential correlation between the CF Abs and persistence of
R.prowazekii in humans.
Rickettsia conorii antigen presentation to CD8 T-lymphocytes by a
murine endothelial cell line.
Marcela Diaz, Hui-Min Feng, and David H. Walker
University of Texas Medical Branch, Galveston, Texas
The ability of murine endothelial cells (SVEC) to effectively process and present
Rickettsia conorii antigens to CD8 T-lymphocytes was investigated. The effect of
Rickettsia conorii infection on MCH class I expression was determined by incubation
with FITC-stained monoclonal antibodies against H-2Kk molecules and by flow
cytometry detection of fluorescence. MHC class I expression was not affected by
Rickettsia conorii infection. The CD8 T-lymphocyte response to antigens presented by
SVEC cells was determined by IFN- production. Immune and non-immune CD8 Tlymphocytes were evaluated. Immune CD8 T-lymphocytes were isolated from the
spleens of mice previously challenged with a sublethal dose of Rickettsia conorii. Nonimmune CD8 T-lymphocytes were isolated from naive mice. IFN- levels were higher
in the supernatant of Rickettsia conorii-infected SVEC cells incubated with immune
CD8 T-lymphocytes than in the supernatant of infected SVEC cells incubated with
non-immune CD8 T-lymphocytes, and that of uninfected SVEC cells incubated with
either immune or non-immune CD8 T-lymphocytes. In conclusion, SVEC cells are
able to effectively process and present Rickettsia conorii antigens to CD8 Tlymphocytes; only immune CD8 T-lymphocytes are able to recognize these rickettsial
antigens.
- 26 -
63
Reactivity of autoimmune sera with antigens in ehrlichial
preparations.
84
Paxton, H., B. Hanson, M. Simanowith, B. Belcher, J. Antony, and W. Liu.
Integrated Diagnostics, Inc., Baltimore, MD, USA
IFA testing for antibodies to intracellular organisms such as ehrlichiae can be
complicated by the presence of autoantibodies in the sera, since considerable
expertise is required to see organism-specific, intracellular fluorescence through
intense host cell antigen staining. This can be especially problematic when screening
serum collections with a high prevalence of autoantibodies, such as we found in
retrospective serum panels collected in malaria-endemic regions. For this and other
reasons, IFA results are often confirmed by Western blotting. While evaluating our
IFA results, however, we found that, unlike other control sera, several ehrlichia IFAnegative, autoimmune sera Western blotted antigens from ehrlichiae purified by
standard Renografin gradient procedures. Several autoimmune sera produced
predominant bands with approximated molecular weights of either 40- to 45-kDa or
27- to 30-kDa. This was of obvious concern since these profiles overlap those of the
specific outer membrane (OM) antigens for HGE and Ehrlichia chaffeensis,
respectively. Similar patterns seen with unpurified, uninfected host cell antigens could
be removed by ehrlichia purification techniques, but the ehrlichia antigen preparations
continued to react with autoimmune sera even after treating with Sarkosyl to produce
ehrlichial OM. Attempts to identify the source of the antigens reactive with the
autoimmune sera, as well as the autoimmunogens involved, are ongoing. In addition
to indicating the potential importance of crossreacting or contaminating antigens,
these studies also demonstrate the desirability of determining the autoimmune status
of a test serum before interpreting Western blots with ehrlichial antigens. Supported
by Dept. of the Army Contract DAMD 17-96-C-6027.
Reception of a combined inactivated typhous and study of its
efficiency
A.N. Pantyukina, W.F. Petrov, T.S. Kovaljova, O.U. Sosnina
Research-and-production assotition «Biomed», Perm, 614089, Russia
The efficiency of typhous vaccine depends that as far as immunodominant
antigenes of the microorganism are full submitted in it.It is known, that
immunodominant antigenic structures are found both in an outside surface rickettsial
cells and on their membranes (membrane-connected proteins). Proceeding from this
fact with the purpose of maintenance of high protective immunity superficial and
membrane connected conservative) components of a rickettsial cells must be
included in a vaccine.From these positions combined inactivated typhous vaccine was
designed. It consisted of two components : purified corpuscular antigene R.
prowazekii, strain Breinl with preserved superficial proteins and soluble fraction,
containing membrane immunodominant structures of rickettsiae.On estimation of
efficiency of this typhous vaccine CITV if was established that it causes 100%
serumconversion on experimental animals with the prevalence of antibodies to R.
prowazekii. CITV provides protection of animals from typhous disease by introduction
of a virulent material in a doze 1:1000 ID 50. It is shown low reactogenicity and
absence of toxicity.The received data open prospects for the use of a given vaccine
with the purpose of formation of protective immunity against typhus.
- 27 -
85A
Diagnosticum for revealing of species-specific antibodies to R.
prowazekii , R. typhi, R. sibirica.
85B
A. N. Pantyukina, W. F. Petrov, T. S. Kovaljova, A. I. Komarova .
Research and production assotiation «Biomed», Perm, 614089, Russia; - Gamaleya
Research Institute for Epidemiology and Microbiology, Moscow, Russia.
The identification of antibodies to R. prowazekii, R. typhi, R. sibirica is based on
the parallel titration of sera with homologous and heterologous antigenes in CFT. The
interpretation of results is carried out on the basis of two of more multiple prevalence
in CFT of the titer of antibodies with homologous antigenes above the titer of
antibodies with heterologous antigenes of the given group. Our attempts to use
another serological tests for the intragroup differentiation were complited with negative
results. It was connected with the presence of group - specific(GSA) and species specific antigenes (SSA) in the preparations. For the reception of SSA of
R.prowazekii, R.typhi, R.sibirica were subjected by the treatment with triton Õ -100.
The GSA were removed by means of excluding immunosorbtion from the received
solubilizates. The prepared SSA are used for designing appropriate erythrocite
diagnosticums for indi rect reaction of hemagglutination or test-system for ensym
immunoassay. The revealing of antibodies has been carried out in sera of the patients
and experimentally infected animals as with typical diagnosticums as species specific diagnosticums. It is shown that with the help of SSA the appropriate to them
antibodies were determinid in sera in both IRHA and ELISA in titer from 1:1000 to up
1:100000 were revealed in sera only. Thus, the received finding open prospects of
absolute intragroup of rickettsioses differentation of the typhous group and the tick
spotty fever group. The application of species - specific diagnostics in practice will
allow to determine the true reason of disease and to nominate correct treatment.
Identification and characterization of linear epitopes recognized
by mouse monoclonal antibodies on the surface protein antigen
of Rickettsia prowazekii.
W.-M. Ching1,2, H. Wang1, G. A. Dasch1.
Naval Medical Research Center, Bethesda, MD, USA1, Uniformed Services
University of the Health Sciences, Bethesda, MD, USA 2
The surface protein antigens (SPAs) of typhus rickettsiae are the outermost
components of their cell envelopes. They are responsible for species-specific
serological reactions and elicit protective immune responses against typhus group
rickettsiae. Mouse monoclonal antibodies were generated to study the
immunochemistry of these proteins. Their epitopes were first localized on the CNBr
fragments of SPAs from typhus group rickettsiae and then identified by ELISA testing
of overlapping decapeptides encompassing the corresponding CNBr fragments. Three
monoclonal antibodies, P6, P7, P8, generated against Rickettsia prowazekii,
recognized fragment PE corresponding to the N-terminus of SPA. P7 cross reacted
with N-terminal fragment T4b of R. typhi SPA and P6 showed weak interaction with
fragment PD of R. prowazekii.. The peptide epitope of P8 was identified as
MGAAMQYNRT (amino acid 1-10) by pepscan. Residues of this peptide required P8
binding were characterized with modified peptides. Substitution of glycine for M1, A4,
and Q6 essentially abolished the binding activity of P8 to this epitope, while
substitution of glycine for N8 and T10 actually increased P8 binding, suggesting that a
decrease in steric hindrance favored stronger antibody binding. The N-terminal 1/3rd
of SPA may present important surfaces to which antibodies can bind. If correct, this
region of SPA may be essential for inclusion in recombinant antigens intended for use
in serodiagnostic tests and vaccines.
- 28 -
96
Study of dynamics of synthesis of TNF and manifestation of NKactivity in C3HA mice infected with Coxiella burnetii or immunized
with inactivated whole cells of C.burnetii (WC-1).
110
Tokarevich N.K., Freidlin I.S., Pogodina O.N., Prokopyeva E.D., Prokopyev A.A
Pasteur Institute of Epidemiology and Microbiology, Institute of Experimental Medicine,
Institute of Cytology, Research Institute of Highly Pure Biopreparations,St. Petersburg.
Infection of C3HA mice with C.burnetii induced an production of TNF after an
intravenous injection of LPS or WC-1.Administration of LPS produced appearance of
noticable amounts of TNF in the serum of these mice in 3 days after their infection with
C.burnetii. The maximum TNF levels were achieved in 5-7 days and remained
practically constant until 14 days. Administration of WC-1 caused maximum TNF
production in 3 days after infection of the animals with C.burnetii. The 2nd rise in the
TNT level was recorded in 14 days. Two peaks of the NK-activity of splenocytes were
revealed, the 2nd peak depending more on the presence of macrophages, i.e., it might
have been due to the TNF stimulation. Preliminary immunization with WC-1 of the
mice infected with C. burnetii enhanced the LPS- and WC-1-dependent production of
TNF. Thus the defensive effect of immunization with WC-1 seems to be due to an
increase in the ability of macrophages to respond with the more intensive TNF
production to the subsequent infection of the animals with C.burnetii.
Testing of inactivated combinated phase I Coxiella burnetii
vaccine in volunteers
Vasilenko A.Z.1, Fatalieva S.F.2, Yablonskaya V.A.2, Kartzeva N.A.3, Misnikov
O.P.1, Tokarevich N.K.3, Tarasevich I.V.2
Military Medicine Research Institute (Sankt-Petersburg)1, Gamaleya Research of
Epidemiology & Microbiology Institute (Moscow) 2, Pasteur Research of Epidemiology
& Microbiology Institute (Sankt-Petersburg)3
A inactivated combinated phase I Coxiella burnetii vaccine (strain Luga-1)
developed at Gamaleya Research of Epidemiology & Microbiology Institute and
Pasteur Research of Epidemiology & Microbiology Institute, Russia was evaluated for
safety and immunogenicity by administering 0,5 ml subcutaneously to twenty
volunteers. The vaccine was safe; there were no serious adverse reacti- ons.
Fourteen recipients experienced no or mild side effects. Five recipients reported mild
headache, myalgia and fever on 1-3 separate days (maximum temperature = 37.138.2 degrees C). One volun- teer, who had a long contacts with C.burnetii,
experienced generalized weakness with fever (maximum temperature = 38.7°C)
lasting less than 24 h. The vaccine was highly immunogenic; all recipients developed
antibody or cell-mediated immunity.
- 29 -
134
Mutagenesis of the invasion-associated locus B gene of Bartonella
bacilliformis.
146C
S.A. Coleman and M.F. Minnick
The University of Montana, Missoula, MT.
B. bacilliformis is a hemotrophic bacterial pathogen that infects human
erythrocytes. The invasion-associated locus A and B genes (ialAB) can confer an
erythrocyte-invasive phenotype upon minimallt invasive strains of E. coli. The ORF for
ialB is 558 bases and encodes a 186 amino acid protein with a predicted Mr of 19,900.
At the amino acid level, iaIB is approximately 60% similar to the virulence
determinants Ail (Adhesion and invasion locus) of Yersinia enterocolitica and Rck
(Resistance to complement killing) of Salmonella typhimurium. The ialB protein of B.
bacilliformis has a putative 22 amino acid secretory signal sequence and is
hypothesized to be exported to the cell surface where it functions as an invasion
factor. To ascertain the hypothesized role of ialB in virulence, ialB- mutants were
created by allelic exchange with a suicide plasmid containing an internal fragment of
IalB.. Homologous recombination between the single chromosomal copy of ialB and
the suicide plasmid led to disruption of the ialB gene which was confirmed using PCR.
Immunoblots developed with antiserum generated against an IalB fusion protein were
used to show that IalB expression was abrogated in the mutant strain. To confirm
IalB's putative role in virulence, ialB- mutants were complemented in trans with a
replicating plasmid containing the full-length ialB gene. PCR confirmed that
complementation rather than homologous recombination occurred. The resulting
strains are currently being used in human erythrocyte adhesion and invasion assays
to assess the role of IalB in B. bacilliformis pathogenicity.
The Rickettsia australis outer membrane proteins A and B.
John Stenos* and David Walker.
Australian Rickettsial Reference Laboratory, Geelong Hospital, Victoria, Australia and
the University of Texas Medical Branch, Galveston, Texas, USA.
The rickettsial outer membrane protein A (rOmpA) has been a distinguishing
feature of the spotted fever group (SFG) rickettsiae whereas the rOmpB is present in
both SFG and typhus group rickettsiae. However, there are members of the SFG,
which have not as yet been shown genetically to contain either of these genes. Using
primers designed to conserved areas of the R. rickettsii rompA a segment of the
homologous gene was amplified from the R. australis DNA. The rest of this gene was
amplified utilising an inverse PCR method. The R. australis rompA is characterised
by an ORF which spans 6318 bp which contains 8 repeat units, 252 bp in length and
one partially repeated unit of 62 bp. This rompA shares close homology with the other
rompA genes but is dramatically different in the repeat region. Conversely, the R.
australis rompB was amplified with conserved primers and displayed close
homologies with the other SFG rickettsiae.
- 30 -
151
Pathological features of Q fever hepatitis
H. Lepidi, D. Raoult
Laboratoire d'Histologie et Unité des Rickettsies CNRS-UPRES A 6020, Faculté de
Médecine de Marseille, France
152
Q fever is a worldwide zoonosis caused by Coxiella burnetii, an obligatory
intracellular organism. The most common acute clinical manifestations include limited
febrile illness, pneumonia, and granulomatous hepatitis.
A wide range of pathological findings have been noted by several pathologists
during Q fever in the liver, bone marrow, and spleen. The tissue damages of Q fever
hepatitis can be divided as two patterns. In the first one, typical lesions consist of a
granuloma with a central clear space (characteristic central "doughnut" hole)
surrounded by peripheral epithelioid macrophages and lymphocytes with often contain
neutrophils and a fibrin-ring. In the latter, non-specific granulomas show a central
clear space (lipogranulomas). Other non-vacuolated granulomas are nondistinctive
and are composed of histiocytes admixed with neutrophils, mononuclear inflammatory
cells, and variable numbers of giant cells. Moreover, several nongranulomatous
histological changes are seen, frequently including steatosis and nonspecific reactive
hepatitis visualized as portal mononuclear inflammatory infiltrates.
However, no pathological features are specific of Q fever hepatitis and, even for
highly suggestive pictures such as the fibrin-ring granuloma itself, numerous
differential diagnosis must be considered as granulomatous diseases. The diagnosis
of Q fever remains today established by serologic procedures.
In most cases, no organisms are identified in liver biopsies. It is not surprising
since biopsies are usually performed several days to weeks after the onset of the
disease and the microorganisms are probably degraded.
IL-12 potentiates Th1-type responses in human boutonneuse fever
S. Milano, P. D'Agostino, G. Di Bella, M. La Rosa, C. Barbera, V. Ferlazzo, R.
Caruso, P. Mansueto, G.B. Brini, A. Barera, G. Vitale, S. Mansueto, E. Cillari
Institute of Internal Medicine and Institute of General Pathology, University of
Palermo, Faculty of Medicine, Palermo, Italy
Since interleukin (IL)-12 contributes to the resistance to intracellular pathogens,
this study was undertaken to examine the role of IL-12 in the regulation of immune
response against Rickettsia conorii in 20 Sicilian patients with boutonneuse fever
(BF). Data indicate that peripheral blood mononuclear cells (PBMC) from acute BF
patients were able to produce IL-12 in response to in vitro stimulation with rickettsial
antigen; this production was higher than that detected in healed patients. On the
contrary, IL-12 serum levels were undetectable, probably because endogenous IL-12
is produced very early in the response to infectious agents.
IL-12 secretion by PBMC from BF patients was potentiated by recombinant
interferon gamma (IFN-) or anti-IL-10 monoclonal antibodies (mAb). Firtehrmore, the
treatment with anti-IL-12 mAb reduced IFN- synthesis. These results indicate that
treatment of acute BF patients with IL-12 shifted the response toward a Th1-type
response, IL-12 and IFN- are interdependant and they may be associated with the
immunity against rickettsias.
- 31 -
154A
Phagocytosis impairment by C. burnetii and CR3 activity
A. Moynault, S. Meconi, D. Raoult, C. Capo, J. L. Mege
Unité des Rickettsies, Faculté de Médecine, 27 Bd J. Moulin, 13385 Marseille Cedex
05, France
196
We previously demonstrated that virulent C. burnetti organisms are poorly
phagocytosed by human monocytes whereas avirulent variants are efficiently
phagocytosed. The v3 integrin is involved in the uptake of virulent and avirulent
bacteria while CR3, a 2 integrin (CD11b/CD18) known to be a phagocytic receptor, is
necessary for the high phagocytosis level of avirulent organisms. We hypothesize that
virulent C. burnetii organisms down-modulate CR3 activity. The preincubation of
THP1 monocytes with virulent bacteria reduced the ingestion of unopsonized
zymosan (involving lectin sites of CR3) without altering the uptake of iC3b-coated
zymosan (involving iC3b sites of CR3) or IgG opsonized erythrocytes (involving FcR).
This defect in CR3 activity was not due to a lower expression of membrane CR3
assessed by flow cytometry. It may be related to cytoskeletal changes since virulent
C. burnetti organisms but not avirulent bacteria stimulate actin reorganization. We
developed a new methodology to simultaneously study actin filaments (using bodipy
phallacidin) and CR3 activity. Polystyrene particles covered by specific antibodies
(such as CD18) were incubated with monocytes and the particle-monocyte interaction
quantified. In the presence of virulent C. burnetii, this interaction was dramatically
decreased in monocytes which reorganized their actin cytoskeleton. A colocalisation
study by confocal microscopy also showed that polystyrene particles which remained
bound to monocytes stimulated by virulent bacteria were excluded from areas rich in
filamentous actin and cell deformations. It is concluded that the functional impairment
of CR3 induced by virulent C. burnetti is related to cytoskeletal changes.
Role of leukocyte response integrin, integrin-associated protein
and CR3 in phagocytosis of Coxiella burnetii by monocytes
S. Meconi*, C. Capo*, F. P. Lindberg§, D. Raoult*, E. J. Brown§, and J. L. Mege*
* Unité des Rickettsies, Faculté de Médecine, Marseille, FRANCE
§ Washington
University in St. Louis, MO 63110-1093, USA.
Several pathogens exploit macrophages as a niche for survival and replication.
The success of this strategy requires the subversion or the avoidance of microbicidal
functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly
intracellular bacterium which multiplies in myeloid cells. Its survival may depend on
the selective use of macrophage receptors. The uptake of virulent C. burnetii by
monocytes was significantly lower than that of avirulent variants. We found that a mAb
specific for the Leukocyte Response Integrin (LRI) largely inhibited the phagocytosis
of virulent and avirulent bacteria. Hexapeptides KGAGDV and KGRGDV, known to
prevent LRI-dependent adhesion, also blocked the uptake of bacteria. KGAGDV was
more effective than KGRGDV in inhibiting the phagocytosis of virulent C. burnetii
whereas both peptides were equally effective in inhibiting the ingestion of avirulent
variants. These findings emphasize the specific role of LRI in the phagocytosis of
virulent C. burnetii. mAb recognizing Integrin-Associated Protein (IAP) also blocked
phagocytosis of virulent and avirulent C. burnetii. In contrast, antibodies to CR3
blocked the efficient phagocytosis of avirulent bacteria, but had no effect on the
limited ingestion of virulent organisms. Uptake of avirulent C. burnetii was also
markedly depressed in macrophages from IAP-deficient mice, while the uptake of
virulent bacteria was unaffected by IAP deficiency. These data suggest that avirulent
C. burnetii were phagocytosed through a mechanism involving engagement of
LRI/IAP and activated CR3, but that virulent bacteria limit phagocytosis by preventing
CR3 activation.
- 32 -
197B
Effect of interleukin-10 on Coxiella burnetii replication in human
monocytes
198
E. Ghigo, J. Dellacasagrande, C. Capo, D. Raoult, J. L. Mege
Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Marseille, France
Coxiella burnetii, an obligate intracellular microorganism infecting myeloid cells,
is the agent of Q fever. Q fever endocarditis is characterized by defective cellmediated immunity and the overproduction of interleukin-10 (IL-10) and Transforming
Growth Factor-1 (TGF-1) by monocytes. We hypothesize that immunoregulatory
cytokines including IL-10, IL-4 and TGFC. burnetii in human
monocytes. Monocytes were infected with C. burnetii and intracellular bacteria were
revealed by Gimenez staining or indirect immunofluorescence. Bacterial viability was
also determined by replication in HEL permissive cells. In control monocytes, C.
burnetii survived for 12 days but did not replicate. When monocytes were pretreated
by IL-10, the initial infection of monocytes was increased and bacterial replication
steadily increased to reach a maximun after 6 days. The addition of IL-4 or TGFhad no effect on C. burnetii infection of monocytes. When IL-10 was added to
monocytes already infected by C. burnetii, the bacterial replication to monocyte
increased as in pretreatment experiments. IL-10-mediated replication of C. burnetii did
not involve reactive oxygen intermediates or the production of IL-1 receptor
antagonist. In contrast, Tumor Necrosis Factor (TNF) synthesis determined by RTPCR and TNF secretion detected by ELISA were completely inhibited by the
incubation of monocytes with IL-10. The role of TNF in the replication of C. burnetii
was confirmed by adding exogenous TNF to infected monocytes treated by IL-10,
which partly reversed the effect of IL-10. This study demonstrates that only IL-10
enables C. burnetii replication in human monocytes by inhibiting TNF synthesis at the
transcriptional level.
Coxiella burnetii killing is associated with apoptosis of THP1
monocytes
J. Dellacasagrande, C. Capo, D. Raoult, J. L. Mege
Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Marseille, France
The treatment of infectious diseases caused by intracellular bacteria, such as Q
fever, may benefit from cytokines acting on macrophages. Monocytic THP1 cells were
infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with
interferon- (IFN-). While C. burnetii multiplied in untreated monocytes, IFN- reduced
bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h.
IFN- also affected the viability of infected cells. Cell death resulted from apoptosis;
occurring 24 h after the addition of IFN-, it reached a maximum after 48 h and was
followed by necrosis. Reactive oxygen intermediates were not required for C. burnetii
killing since monocytes from patients with chronic granulomatous disease were
microbicidal in response to IFN-. The role of cytokines was also investigated. IFN-
elicited a moderate release of interleukin (IL)-1 in infected monocytes. Moreover, the
IL-1 receptor antagonist did not affect C. burnetii survival, suggesting that IL-1 was
not involved in the bacterial killing induced by IFN-. Tumor necrosis factor- (TNF)
was involved in IFN--induced killing of C. burnetii and cell death. IFN- induced
mRNA expression and sustained secretion of TNF. Neutralizing antibodies to TNF,
as well as antibodies directed against TNF-RI and TNF-RII, significantly prevented
IFN--dependent killing of C. burnetii and cell death. These results suggest that IFN-
promotes the killing of C. burnetii in monocytes through an apoptotic mechanism
mediated in part by TNF.
- 33 -
200A
Mechanism of Coxiella burnetii-stimulated production of tumor
necrosis factor by monocytes
200B
J. Dellacasagrande*, E. Ghigo*, S. Machergui*, D. Raoult*, R. Toman §, C. Capo*,
J. L. Mege*
1 Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Marseille,
France - § Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak
Republic
We previously showed that monocytes from patients with Q fever endocarditis
spontaneously produced Tumor Necrosis Factor (TNF). Our aim was to study the
production of TNF in response to C. burnetii. THP1 monocytes were stimulated with
C. burnetii: TNF mRNA production was assessed by RT-PCR, protein levels were
measured by ELISA and TNF activity was determined using a specific bioassay. A
two-hour contact between THP1 cells and C. burnetii was sufficient to induce maximal
levels of TNF mRNA. Thereafter, immunoreactive and bioactive TNF were secreted
by THP1 monocytes with a maximum after 6-12 hours of stimulation with C. burnetii.
TNF production did not require bacterial phagocytosis since cytochalasin D did not
reduce the expression of TNF mRNA. The interaction of C. burnetii with monocytes
via v3 integrin is necessary to TNF synthesis because antibodies specific for v3
integrin or inhibitory peptides containing RGD sequences strongly diminished the
expression of TNF transcripts. On the other hand, polymyxin B, an antibiotic which
inhibits LPS activity, reduced the expression of TNF transcripts induced by C. burnetii.
In addition, purified LPS of C. burnetii induced TNF mRNA. We postulate that TNF
production by monocytes stimulated by C. burnetii needs two steps : first, the
attachment of the bacteria to monocytes (via v3 integrin) and, second, a signal
delivered by bacterial LPS.
A murine model of systemic Bartonella henselae infection in
immunocompetent and immunocompromised hosts
E. Bernit, G. E. Grau, P. Brouqui, I. Garcia°, H. Borghi, H. Lepidi and D. Raoult
Unité des Rickettsies, Faculté de Médecine, CNRS UPRES-A 6020, 13385 Marseille,
France
and (°) Dept. of Pathology, University of Geneva, CH-1211 Geneva 4, Switzerland
The clinical outcome of human Bartonella henselae infection is mainly
determined by the immunological status of the host. Cat scratch disease occurs in
immunocompetent individuals, whereas bacillary peliosis and bacillary angiomatosis
are reported in immunocompromised hosts. In order to understand better the
pathophysiology of B. henselae infections, we set up an experimental model of
systemic infection in immunocompetent or immunocompromised (cyclophosphamidetreated) BALB/c mice, and in TNF-R1 transgenic mice. Serology, blood and organ
culture, histopathology and immunochemistry on liver and spleen sections were
performed.
Infected mice showed moderate and transient signs of morbidity. B. henselaespecific IgG antibodies were detected in infected immunocompetent and transgenic
mice. No vascular lesion, particularly peliosis and neoangiogenesis, was observed.
Granuloma size and number did not depend on the size of bacterial inoculum. B.
henselae were revealed by immunofluorescence early after infection in liver sinusoids
and, later on, within granuloma cells. The liver granulomatous response was
significantly reduced in cyclophosphamide-treated mice and in mice expressing high
levels of the TNF-R1 transgene, compared to infected immunocompetent or
transgenic mice expressing low levels of TNF-R1. This model may thus be useful to
study the pathophysiology of B. henselae infection and the role of the host immune
response.
- 34 -
201
An experimental model for Q fever during pregnancy
Andreas Stein,1 Hubert Lepidi,1,2 Jean Louis Mege,1 Thomas J. Marrie,1,3 and
Didier Raoult1*
Unité des Rickettsies, CNRS UPRES-A 6020, Faculté de Médecine,1 and Laboratoire
d'Anatomie Pathologique et de Neuropathologie,2 Centre Hospitalier Universitaire,
13385 Marseille, Françe; Department of Medecine, Dalhousie University and Victoria
General Hospital, Halifax, Canada3
Q-fever is a widespread zoonosis caused by Coxiella burnetii, an obligate
intracellular bacterium which man usually acquires through the inhalation of infected
dust from subclinically infected animals. Although this highly virulent organism is most
concentrated in mammals during parturition, there are few reports on the
manifestations of perinatal Q fever in the human and animal host. We investigated the
affinity of C. burnetii to pregnancy and its abortifacient potential in a murine animal
model. Intraperitoneal infection of female BALB/c mice with C. burnetii, followed by
repeated pregnancies over a 2-year period, resulted in persistent infection associated
to abortion and perinatal death, with a statistically significative decrease in viable
offspring. C. burnetii antigen and DNA were detected in the aborted fetuses, stillborn
mice, and organs of the female mice autopsied at the end of the experiment. In
addition, endocarditis occurred in two of the adult animals and C. burnetii antigen and
DNA were detected in their heart valves. Taken together, our results demonstrate the
abortifacient potential of C. burnetii and the increased risk of persistent infection and
endocarditis in pregnant mice. We suggest that a decline in cellular immunity during
pregnancy may play a role in the development of C. burnetii-associated lesions in the
placenta and endocardium and thus may account for severity and persistence of the
infection.
- 35 -
205
Monday June 14th
Slide session 2
Physiopathology and Immunity
Human Macrophages and Hepatocytes.
David H. Walker, M.D. Patricia Crocquet-Valdes, M.S., and Hui-Min Feng, M.D
University of Texas Medical Branch, Department of Pathology, WHO Collaborating
Center for Tropical Diseases, Galveston, Texas.
Rickettsiae are cleared from the infected host principally by intracellular killing.
Cytokine-activated mouse endothelial cells and macrophages inhibit the growth of
intracellular rickettsiae by an inducible nitric oxide synthesis (iNOS) -dependent
mechanism. Human endothelial cells inhibit intracellular rickettsial growth by a
combination of cytokine-activated tryptophan-depletion and reactive oxygen species.
Because macrophages and hepatocytes appear to be targets in some rickettsial
infections, killing of Rickettsia akari was studied in human hepatocytes (AKN-1 cells)
and macrophages (THP-1 cells) after activation with RANTES and/or cytokines.
Rickettsiae were demonstrated to be killed by iNOS dependent mechanism for the
first time in human cells, hepatocytes activated with RANTES. Human macrophages
activated by RANTES and/or cytokines also inhibited the growth of R. akari by
mechanisms mediated by H2O2 and possibly also nitric oxide. Activated hepatocytes
expressed mRNA of iNOS and the tryptophan-degradation enzyme, indoleamine 2,3dioxygenase (IDO); activated macrophages expressed mRNA of IDO.
- 36 -
159A
The surface protein antigen of Rickettsia typhi: in vitro and in vivo
immunogenicity and protective efficacy in mice
G. A. Dasch1, A. L. Bourgeois1, and F. M. Rollwagen2
92
Naval Medical Research Center, Bethesda, MD, USA 1 and Uniformed Services
University of the Health Sciences, Bethesda, MD, USA 2.
The immunogenicity of water soluble, surface protein antigen (SPA or rOmpB)
purified from Rickettsia typhi was evaluated in two model systems. Splenocyte
suspension cultures from both naive and mice infected with R. typhi were stimulated
in Mishell-Dutton medium with either SPA or sonicated whole cell extracts (TOT) for
up to seven days. With either antigen only typhus-specific IgM was secreted by 4 days
of culture of naive splenocytes and small amounts of IgG3 were detected by 7 days.
Immune splenocytes secreted larger amounts of IgM as well as all four IgG
subclasses beginning on day 3 of culture. TOT antigen was mitogenic while SPA
elicited specific proliferative responses only with immune lymphocytes. High doses of
TOT antigen, and to a lesser extent SPA, suppressed both antibody secretion and
lymphocyte proliferation. In vitro antibody synthesis could be detected with as little as
50 pg of antigen per culture. 0.5-50 µg of soluble SPA elicited dose-dependent
antibody responses in vivo. SPA also protected mice against lethal challengewith R.
typhi. A two dose regimen of 5 µg + 2 µg of soluble SPA or a single 2 µg dose of SPA
in Freund’s incomplete adjuvant was equally protective. These two model systems
should permit further analysis of the structural features of SPA that promote its
immunogenicity and facilitate efforts to develop typhus vaccines suitable for human
use.
148
Abstract not available
- 37 -
Experimental infection of cats and dogs with Bartonella isolated
from domestic and wild carnivores.
1
2
1
1
41
1
Bruno B. Chomel , R. W Ermel , R. W. Kasten , K. Yamamoto , C. C. Chang , R.
Heller3, D. Weber4, A. Poland4, Y. Piemont and N.C. Pedersen4.
1.Department of Population Health and Reproduction, School of Veterinary Medicine,
University of California, Davis, CA 95616, USA; 2. Department of Veterinary
Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station,
TX 77843-4467, USA; 3. Institut de Bactériologie, Université Louis Pasteur, 67000,
Strasbourg, France; 4. Center for Companion Animal Health, School of Veterinary
Medicine, University of California, Davis, CA 95616, USA.
Cats are the principal reservoir of Bartonella henselae, the main agent of cat scratch
disease (CSD), and of B. clarridgeiae, another possible agent of CSD. Dogs are infected with B.
vinsonii subsp. berkhoffii, causing endocarditis, myocarditis and arrhythmias. We have isolated
in the few last years Bartonella species from pumas, bobcats and coyotes from California.
Isolates from pumas and bobcats are close to, but different from B. henselae. From coyotes, we
isolated several strains of B. vinsonii subsp. berkhoffii and a B. clarridgeiae-like strain. Specific
pathogen free cats were experimentally infected with: a) B. vinsonii subsp. berkhoffii isolated
from a coyote (2 cats); b) a puma Bartonella isolate (9 cats); and c) the B. clarridgeiae-like
strain (4 cats). Conventional, seronegative and abacteremic dogs were inoculated with: a)
Bartonella henselae (6 dogs); b) B. vinsonii subsp. berkhoffii isolated from a coyote (2 dogs),
and c) the coyote B. clarridgeiae-like strain (2 dogs). None of the 6 dogs inoculated with B.
henselae became bacteremic. However, one of the 2 dogs receiving a 10 9 CFU/ml inoculum
had one positive colony in one instance 14 days after inoculation. None of the two cats infected
with B. vinsonii subsp. berkhoffii became bacteremic. Conversely, all 4 dogs inoculated with the
two coyote strains and all 9 cats inoculated with the puma strain became bacteremic. Three of
the 4 cats infected with the coyote B. clarridgeiae-like strain became bacteremic. Bartonella
henselae seems to be highly associated with its feline reservoir, whereas the B. clarridgeiae-like
strain appears to be able to infect both canids and felines.
Molecular heterogeneity of the 28 kDa surface antigen multigene locus of
Ehrlichia chaffeensis isolates suggests that it plays a role in immune evasion.
G. Roman Reddy
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, KS 66506
Ehrlichial infections including those by Ehrlichia chaffeensis, E. canis, Cowdria
ruminantium, and Anaplasma marginale persist in mammalian hosts. Persistent
infections are advantageous to pathogens because they serve as stable reservoirs of
infection, thereby increasing the chance of tick transmission, and complicate the
development of effective control measures. Earlier, we described the characterization
of multigene locus encoding 28 kDa surface antigens from three closely related
Ehrlichiae; E. chaffeensis, E. canis and C. ruminantium. The presence of only three
immunogenic regions per gene that are variable in different gene copies, together with
the presence of only one transcriptionally active gene, suggests a mechanism of
immune evasion in these Ehrlichiae (G. R. Reddy et al. 1998, Biochem Biophys Res
Commun, 247, 636-643). In this study, immunological specificity of the 28 kDa
antigens is mapped using recombinant proteins and monoclonal antibodies. The 28
kDa gene loci for two E. chaffeensis isolates (Jax and St. Vincent) have also been
characterized and compared with the E. chaffeensis Arkansas isolate. The transcripts
encoded by these isolates are different and are not identical to any of the cloned
genes of Arkansas isolate, E. canis or C. ruminantium. DNA filter hybridization
analyses revealed extensive restriction enzyme length polymorphisms among the
isolates. Furthermore, the coding sequences of the 28 kDa genes contain many
features supporting that they undergo recombinations. Molecular heterogeneity in the
gene locus and encoded transcripts in different isolates is an expected outcome when
it undergoes recombinations to produce antigenic variants.
- 38 -
5
Mechanisms of Immunity to Ehrlichia chaffeensis in Mice.
Hui-Min Feng, M.D., and David H. Walker, M.D.
University of Texas Medical Branch, Department of Pathology, WHO Collaborating
Center for Tropical Diseases, Galveston, Texas.
156
The experimental eludication of immune mechanisms against Ehrlichia
chaffeensis was approached by the use of gene knockout mice for the functions of
CD4 T-lymphocytes (MHC class Il), CD8 T-lymphocytes (MHC class I), cytotoxic Tlymphocytes (perforin), nitric oxide synthase (NOS), and gamma interferon (IFN-),
nude mice, SCID mice with adoptive transfer of immune CD4 or CD8 T-lymphocytes
or immune or nonimmune unfractionated lymphocytes, and SCID mice with further
depletion of the function of macrophages, IFN-y, TNF-, or NK celîs. All of the gene
knockout mice were resistant to E. chaffeensis, indicating that CD4 and CD8 Tlymphocyte subsets, perforin, NOS, and IFN- were flot essential as host defenses
against E. chaffeensis. Adoptively transferred immune CD8 T-lymphocytes protected
SCID mice from death. However, nonimmune unfractionated lymphocytes were
capable of reconstructing a protective immune response if given early enough in the
course of infection. Antibodies were not a requirement for resistance to infection.
Interferon gamma dominates the early cytokine response to
murine infection with the agent of Human Granulocytic
Ehrlichiosis.
Mustafa Akkoyunlu, and Erol Fikrig
Yale University School of Medicine, Department of Medicine,
Rheumatology, LCI 604, 333 Cedar St., New Haven, 06520, U.S.A.
Section
of
Cytokine response during murine infection with the HGE agent was scrutinized
in this study. Blood PCR revealed that HGE agent DNA was detectable on day 2. The
PCR signal was most intense on day 8 and then decreased in intensity on day 15.
HGE agent DNA was weakly apparent on days 21, 30 and 45. The percentage of
infected peripheral blood neutrophils was also maximal at day 8. Levels of Th1 (IFN-,
IL-12) and Th2 (IL-4, IL-10) cytokines were measured in ELISA and RT-PCR. Serum
ELISA demonstrated increased levels of IFN-, but not the other cytokines, on day 5.
In vitro stimulation of HGE infected mice splenocytes also induced IFN- secretion.
RT-PCR revealed high IFN- mRNA levels on days 2, 5, 8, 15, and low levels of IL-12
and IL-10 on days 5, 8, and 15. IL-4 mRNA was only detected on day 15. To address
the role of IFN- in the clearance of HGE from blood we infected IFN- knockout (-/-)
mice with the agent of HGE. Both, the IFN- -/- and the wild type BL56 mice had peak
infection on day 5. However, the level of bacteremia was higher in IFN- -/- mice than
the wild type BL56 mice. Despite the difference in the bacterial load on day 5, by day
15, HGE DNA was undetectable in the blood of control and knockout mice. These
results suggest that IFN- has an impact on the suppression of acute infection while
clearance of infection from blood is independent of IFN-.
- 39 -
42
A feline model of granulocytic ehrlichiosis infection with AIDS
Janet Foley
59
In order to evaluate the effect of pre-existing immunosuppression on
ehrlichiosis, and the immunosuppressive effect of ehrlichiosis, a feline coinfection
model using human granulocytic ehrlichiosis and feline AIDS was studied. Cats were
divided into three treatment groups: cats infected with HGE alone, cats infected with
feline immunodeficiency virus (FIV) with clinical AIDS, and cats with AIDS infected
with HGE. Following infection, monitoring included: clinical status and rectal
temperature; complete blood count; CD4+ and CD8+ cell counts by flow cytometry;
serum liver transaminase measurement; testing for antibodies against nuclei, FIV, and
ehrlichia; polymerase chain reaction testing for FIV, ehrlichia, and the cytokines IFN-,
IL-2, IL-4, IL-I O and TNF; and cytology of joint, bone marrow and lymph node
aspirates. Additionally, cats received modified live herpesvirus vaccine and
recombinant feline leukemia virus vaccine on day 10 following ehrlichia inocujation,
and were assessed over three weeks for their ability to make antibodies in response
to the vaccine. The cats were also challenged with an intradermai inoculum of
Yersinia pseudotuberculosis, an intracehular pathogen, and biood and lymph node
cultures were performed after inoculation in order to assess cell-mediated immunity.
Data were analyzed to determine whether AIDS predisposes individuals to more
severe ehrlichiosis and whether there was detectable immunosuppression produced
as a resuit of elirlichiosis.
Cowdria MAP1 protein sequence similarity clustering and cross
protection among isolates
M. T. E. P. Allsopp', C. M. Hattingh1, J. C. Maillard2, A. Bensaid3, I. Chantal2 & B.
A. Allsopp'
1Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa - 2CIRADEMVT, Montpellier, France - 3CIRAD-EMVT, Pointe-à-Pitre, Guadeloupe
In South Africa, the rickettsial haemoparasite Cowdria ruminantium is
transmitted by the tick Amblyomma hebraeum and clinical heartwater cases are
associated with the distribution of this tick. It is one of the three most important tickborne diseases in Africa and since much of South Africa is unsuitable for the
cultivation of crops, animal husbandry is essential to the survival of many
communities. Development of inactivated or recombinant vaccines against the
disease is therefore important.
Different Cowdria genotypes with differing immunogenicities exist in the field
and it is possible that some of these may represent different rickettsial species. Before
any large-scale vaccination programme can be carried out it will be necessary to
obtain detailed information on the distribution of the different variants. In order to
assess the distribution of MAPi immunotypes we have amplified, cloned and
sequenced mapi genes from a wide range of heartwater isolates.
Phylogenetic analysis of the MAP1-derived amino acid sequences from 25
Cowdria isolates indicates that there is no correlation between geographical
distribution and MAP1 immunotype. There are four MAP1 sequence similarity clusters
and limited cross-protection experiments suggest that there may be greater crossprotection between isolates within a cluster than between those in different clusters.
- 40 -
4
Optimization of the MAP1 DNA Vaccine for Sheep
Michael V. Bowie1, Aceme Nyika2, Suman M. Mahan2 and Anthony F. Barbet1.
1Department of Pathobiology, College of Veterinary Medicine, University of Florida,
Gainesville, Florida 32610; 2UF/USAID/SADC Heartwater Research Project, Harare,
Zimbabwe.
7
It was earlier reported that the MAP1 gene of Cowdria ruminantium protected mice
against homologous challenge as a naked DNA vaccine. Because ruminants are natural hosts
of infection, it was important to determine if DNA vaccines could provide protection in sheep.
The present trial was conducted (i) to determine the most appropriate mode of vaccination for
sheep and (ii) to determine if cell-mediated immune responses could be induced using the
MAP1 DNA vaccine in sheep. Twenty-four animals (eight groups) were vaccinated
intramuscularly (IM) and/or intradermally (ID) using conventional needle-injection and/or
needleless jet injection (Biojector®) with and without MAP1 boosts, and with and without
bupivacaine. Isolated peripheral lymphocytes from sheep were stimulated with MAP1 and a
lymphocyte proliferation assay (LPA) was performed. MAP2 and concanavalin A were used as
controls. Weekly serum samples were measured for seroconversion in two enzyme-linkedimmunosorbent assay (ELISA) formats and Western blots. Lymphocytes from sheep immunized
using the Biojector® IM/needle ID, the Biojector® IM/MAP1 boost, and Biojector® IM/needle
ID/MAP1 boost responded when stimulated with MAP1 in the LPA. In a separate study,
proliferating lymphocytes from DNA-vaccinated ± MAP1 boost sheep were shown to be
predominantly CD4+ and CD8+ T cells by FACS analysis. A greater than four log endpoint
antibody titer was measured in groups vaccinated with Biojector® IM and Biojector® IM/needle
ID when followed by a MAP1 boost using both ELISA formats. Reactivity and specificity for the
MAP1 was confirmed on Western blots. The best response, based on lymphocyte proliferation
and seroconversion, resulted from the MAP1 DNA vaccine delivered with the Biojector®
IM/needle ID followed by a MAP1 boost. These data show that priming with DNA vaccine
followed by a recombinant protein boost induces strong immune responses in sheep and
should be considered for delivery of recombinant vaccines against heartwater.
The role of T cells in immunity to Cowdria ruminantium infections.
S.M. Mahan1,3, D. Mwangi1,2, B. Byrom1, A. F. Barbet3 and D. Mckeever2
1 UF/USAID/SADC Heartwater Research Project, Harare, Zimbabwe; 2 ILRI, Nairobi,
Kenya; 3 University of Florida, U S A.
DBA/2 mice and bovines were immunized by the infection and treatment
method with C. ruminantium and the protective immune responses evaluated.
C. ruminantium infections induced TH1 immune responses in the DBA/2 mice
which were sustained in immune mice. T cells from spleen of immunized mice
proliferated specifically to C. ruminantium antigens. High levels of IFN  and IL-2 but
not IL-4, IL-5, IL-6, IL-10 or TNF were detected in supernatants of lymphocyte
proliferation assays. Anti-Thy 1.2 antibody successfully abrogated the immunity that
was induced in these immune mice. Athymic (C57B1/6) mice were more susceptible
to infection than normal mice. CD4 knockout (C57B1/6) mice were more susceptible
than CD8 knockout mice, although both types of mice could be immunized by the
infection and treatment method. These data demonstrate that T H1 cell responses
(CD4+ and CD8+ T cells) have a role to play in immunity to infection.
Peripheral blood mononuclear cells (PBM) from cattle immunized against C.
ruminantium infection proliferated in vitro in the presence of autologous infected
endothelial cells or monocytes. By FACS analysis the responding cells belonged to
the CD4+ and T cell lineages. When examined using RT-PCR, these responding
cells showed high expression of IFN, TNF, TNF, IL2R genes and low expression
of IL-2 and IL-4. The supernatants of proliferating cells contained high levels of IFN.
These data collectively indicate that C. ruminantium infection induces TH1
responses in both mice and cattle. In addition, T cells may contribute to immunity in
cattle.
- 41 -
12B
Importance of antigenic variation in persistence of the Ehrlichia
Anaplasma marginale.
G.H. Palmer, W.C. Brown, D.M. French, T.F. McElwain, T.C. McGuire, D. Stiller,
and F.R. Rurangirwa.
Anaplasma marginale is an ehrlichial pathogen of cattle which establishes
lifelong persistent infection. This persistence is fundamental to continued transmission
as transovarial passage of A. marginale within the tick vector does not occur.
Persistent A. marginale infection is characterized by sequential cycles of rickettsemia,
each composed of a progressive, logarithmic increase in rickettsemia followed by a
precipitous decrease. Each rickettsemic cycle reflects the emergence of A. marginale
bearing structurally and antigenically variant major surface protein 2 (MSP2). This
emergence reflects transcription of polymorphic msp2 genes and results in MSP2
variants typified by amino acid deletions, substitutions, and insertions within a single,
central hypervariable region. Importantly, this hypervariable region is within the
hydrophilic domain of MSP2 and is bound by variant-specific antibodies against
surface epitopes. The B cell epitopes in the variant-specific surface domain are not
recognized by antibody when the A. marginale emerges at the beginning of a
rickettsemic cycle but are recognized by antibody as the cycle terminates. Thus,
persistent rickettsemia is characterized by sequential emergence and replication of A.
marginale expressing MSP2 antigenic variants followed by development of a variant
specific immune response. The presence of MSP2 homologues in Ehrlichia
chaffeensis, E. equi, E. canis, and Cowdria ruminantium suggests that these closely
related pathogens may use similar mechanisms to persistent in their reservoir hosts.
- 42 -
100
Tuesday June 15th
Poster session 3
Emerging Rickettsioses
Influence of Galavit on experimental Astrakhan spotted fever
(ASF).
M.Nelubov,M.Abidov,V.Makarova,A.Milovanov,I.Tarasevich.
The Centre of Modern Medicine,Institute of Humans Morphology,Gamaleya Institute
for Epidemiology and Microbiology
Galavit is antiinflammator and immunomodulator,successfully using for
treatment of some diseases (acute typhoid etc.).The influence of Galavit have been
investigated during experimental ASF. The inbread white mice were infected I/p 10 ID
50 of the agent (strain "A-P-I"). Galavit was injected s/c on 4th -8th days of the
disease. In the same days brain,spleen,liver were taken and smears from peritoneum
were done. The clinical and morphological data in the control group of mice were
typical for experimental ASF. In the result of treatment by Galavit have been marked
more light clinical course and earlier recover, increase of lymphocytes in subepindinal
space,absence of sladge reaction and trombosys,shorten angiopatic phase,increase
macrophages in liver,s viens and portal ways,their imbibition in parenhima,increase of
regenerative reaction of hepatocytes, more rapid regeneration of spleen follicules. In
the conclusion one can say that Galavit may be the perspective preparation for
treatment of Rickettsial diseases.
- 43 -
8
Murine typhus in central tunIsia : A report of 7 cases.
A.Letaïef*, M. Chakroun**, F. Bahri*, N. Bouzouaïa**, L. Jemni*
*Services de Maladies Infectieuses, CHU F. Hached, Sousse. ** CHU F. Bourguiba,
Monastir.
27A
Murine or endemic typhus, caused by Rickettsia typhi, has been reported from
all continents. In the seventies, no case of murine typhus was diagnosed in Tunisia.
Nevertheless, when specific serology was made between 1984 and 1992, 7 cases of
typhus were diagnosed at Sousse (Central Tunisia). In this present work, we report
the clinico-epidemiological characteristics of 7 cases (4M, 3F) of murine typhus
diagnosed in our hospital since 1993.
Diagnosis, clinically unsuspected, was confirnied by systematic serology (5
cases in one year). Besides brutal onset of fever, absence of "tache noire", clinical
characterjstics were : rash (4 cases), prostration (6 cases), meningism (2 cases),
pneumonia (3 cases). There was no predilection of season. Biological findings were
slightly similar to Mediterranean spotted fever (MSF). Clinical diagnosis were MSF (5
cases), Q fever (1 case) and pneumonia (1 case). Serology confirmed all diagnosis
with cross reactivity with R. conorii.
In conclusion, we insist that physicians should be alert for this disease in our
country. More specific studies are needed to evaluate real prevalence and type of
typhus in Tunisia.
40A
Recent studies on Scrub Thyhus in China.
Chen Xiangrui.
Institute of Microbiology & Epidemiology Beijing P. R. China
100071
Scrub Typhus, a common infectious disease in South-eastern China, has
recently spread to North China, including Jiangsu, Liaoning, Jilin, Heilongjiang,
Shanxi, Hebei provinces. In these regions, it generally occurs in late fall and early
winter(from August to December). The serum investigation proves that Karp is the
dominant serotype in South China. The infectious rate of Scub Thyphus in different
foci was about 2.0-68.0 %. Although its mortality is very low, there are some cases die
of misdiagnosis, for example, 5 cases in Shanxi province in 1995. Clinical features of
Scrub Thyphus in China is recognized as similar to the illness described in other
countries.
The rodents in the ecology of Scrub Thyphus are Ratttus flaripectus, Rattus
rettus slanderi and Mus musculus in South China, Apodemus Agrarius in North China;
the reservoir are L. Akamushi, L. Deliensis in South China, L. Scutellaria in North
China.
By PCR/RFLP analysis and 56 kDa type-specific antigen gene sequencing, we
found that the Shanxi strain, which isolated from the patients of the spotted fever in
Shanxi, is different from all standard strains. Now, we are carrying on further studies
of Scrub Thyphus on molecular biology, such as 47 kDa and 56 kDa antigen gene
sequencing, cloning, expression for diagnosis and prevention.
- 44 -
Isolation and primary identification of H-5 strain of Spotted Fever
Group Rickettsia directly form the patient’s blood samples in
Heilongjiang province .
54A
Wu Yimin Wei Anming Hulingmei Liu Xinxin Yang Qing Zhang Zhiqiang Lu
Zhixin,
Institute of Medical Science,PLA,Shenyang,P.R.China.
Spotted fever(SF) was investigated at Suifenhe and Dongning area in
Heilongjiang province in China from May to Jun in 1996.We focus our work on finding
patient and isolation pathogen.We found 7 persons who were bited by ticks and
shown SF clinical symptoms. Antibodies against Spotted Fever Group Rickettsia
(SFGR) in these patient’s sera were detected with micro-IF. And in 4 of 7 samples IgM
and IgG in two sera rose (=4 times), Blood(5-7ml) was drawn during the early stage of
illness and injected into male guinea pig by intraperitoneal. The guinea pig incubated
with H-5 samples shown fever, testis swollen. The embryonated eggs were all death
after incubation 5-7days,and rickettsiosiae stain was positive(++).Compared with
SFGR international strain and R.Heilongjiang 54 strain with mouse serum by microIF,H-5 strain is identical to R.Heilongjiang 54 strain. Both of them have the same
antigen property.190KDa antigen gene of H-5 and Other reference strain were
amplified with a pair of primer (190.70p and 190.602n). The amplified fragment
analyzed with RFLP(Pst I) show that H-5 strain is identical to R Heilongjiangii 54
strain, but differs from R.sibirica 246 strain, R.conorii simko stain and R.rickettsii R
strain signigicantly.H-5 strain was isolated from the patient blood specimen directly
demonstrated that H-5 strain can cause disease in human.
Spotted fever group rickettsioses in China
Fan Ming-yuan * Zhang Jian-zhi * Chen Min * Yu Xue-jie+
*:Department of Rickettsiology, Institute of Microbiology &Epidemiology, Chinese
Academy of Preventive Medicine Beijing 102206 P.R.China + Department of
Pathology, Medical Branch, The University of Texas, Galveston TX77550
Spotted fever rickettsioses are tick- or mite-borne diseases caused by spotted
fever group (SFG) rickettsiae. The diseases are world widely distributed. In China, the
investigation of SFG rickettsiae began in 1958. At present, eighteen strains of SFG
rickettsiae have been isolated frorn patients, ticks, tick ova and rodents. The rickettsial
isolates belong to at least four types of SFG rickettsiae including R.sibirica, Hulin
isolate (HL-93), Innermongolia isolate (Ha-91) and Heilongjiang isolate (HLJ-054)
R.sibirica causes North-Asian tick borne spotted fever. The disease caused by
other rickettsial isolates has not yet been determined. This paper is to review the
history of the Chinese SFG rickettsioses and the current situation about the
epiderniology and ecology of SFG rickettsioses in China.
- 45 -
57
Human Monocytotropic Ehrlichiosis (HME): Epidemiological,
Clinical and Laboratory diagnosis of a newly emergent infection in
the United States.
61
Juan P. Olano1, Edwin Masters2, Louis Cullman3, Wayne Hogrefe3, Xue-Jie Yu1,
and David H. Walker1.
1University of Texas Medical Branch, Galveston, TX, 2Regional Primary Care, Inc.,
Cape Girardeau, MO, 3MRL Diagnostics, Cypress, CA
HME was recognized in the United States in 1987. This study addresses
clinical, epidemiological and laboratory diagnostic issues of HME based on results of
an ongoing prospective study in Cape Girardeau, Missouri. Eighty-two patients were
enrolled in the study. Samples were tested by immunofluorescent assay (IFA), PCR,
and Western immunoblotting (WI). In addition, isolation was attempted in different cell
lines including DH82, HL60 and THP-1 cells.
Twenty-two cases were diagnosed with HME during 1997 and 1998. Eleven
cases were diagnosed by IFA only, three by PCR only, one by serology and
immunohistology and seven by both PCR and IFA. The target genes for amplification
included the 16S rRNA gene, the 120 kDa protein gene and the nadA gene. Ehrlichial
DNA was detected in four samples by the nadA gene and the 16S rRNA gene,
respectively and in six samples by the 120 kDa protein gene. DNA sequence analysis
of the PCR products revealed more than 99% homology with the E. chaffeensis
genes. Seroconversion was documented in seven cases. IFA results were confirmed
by WI . No isolates have been obtained.
Based on our diagnostic definition criteria, we have 17 confirmed cases of
HME and five possible cases. Thus, provisional incidence of HME in Cape Girardeau
is 8 cases per 100,000 population during 1997 and 14 cases during 1998. The 120
kDa protein gene appears to be more sensitive for the diagnosis of HME in the acute
phase. Both PCR and IFA are useful for the accurate diagnosis of HME.
Bartonella henselae as a Causative Agent of CSD: Case Report
Dzelalija B.1, Petrovec,M.2 ,Avzic-Zupanc,T.2
1General Hospital Zadar,Department of Infectious Diseases, Zadar, Croatia - 2Institute
ofMicrobiology and Immunology, Medical Faculty, Ljubljana, Slovenia
It is well known that B.henselae is the main etiological agent of cat scratch disease
(CSD),and is responsible for bacteremia, endocarditis, bacillary angiomatosis, peliosis hepatitis
and neurological disorders. CSD manifests itself primarily by a chronic lymphadenopathy
associated with cutaneous lesions caused by cat scratches or a cat bite. Diagnosis of CSD has
traditionally required the presence of three of four criteria: contact with a cat resulting in a
primary lesion, a positive skin test (or serologic testing for antibodies to B. henselae as a
suitable alternative), regional lymphadenopathy, and the presence of characteristic
histopathologic features. Hereby we present a case report of a 21-years old man from Zadar,
Croatia with clinical picture characteristic of cat scratch disease. Eight days after contact with a
cat, patient was presented with primary inoculation papula and pustula and regional
lymphadenitis. In a period between the originate of inoculation and larged lymph nodes (from 2
to 10 weeks) the patient suffered from weakness, headache, myalgia, arthralgia and moderate
fever. Laboratory parameters were in normal range. The histopathologic findings of affected
lymph nodes include stellate caseating granulomas. The presence of prolonged low-grade fever
and algic syndrome indicated a need for additional diagnostic treatment. By using IFA method,
a seroconversion of specific IgG antibodies and a fourfold rise of IgM antibodies against B.
henselae was detected in paired sera. Although, a ten-days peroral doxycycline (2 x 100 mg)
antibiotic treatment was administered on fourth week of the illness, signs and symptoms were
ceased after three months.
- 46 -
64
Recent findings on Ehrlichia organisms in the Free State province,
South Africa
1
77A
2
A-M Pretorius & PJ Kelly
1 Dept of Medical Microbiology, Faculty of Health Sciences, UOFS, Bloemfontein,
South Africa 2 Faculty of Veterinary Science, University of Zimbabwe, Harare,
Zimbabwe
Ehrlichia canis, etiological agent of canine tropical pancytopenia, that was first
described in dogs from Algeria, has been reported to occur worldwide. In the USA
dogs are regarded as potential reservoirs of E. chaffeensis (causative agent of human
ehrlichiosis) infection as they are susceptible to natural and experimental infection. In
nature, the white-tailed deer (Odocoileus virginianus) has been determined as the
reservoir of E. chaffeensis. It was therefore necessary to determine whether dogs
and ticks collected from dogs and wild animals from game parks in South Africa are
naturally infected with E. chaffeensis and E. canis.
Sera from 161dogs in the Bloemfontein area and 45 sera of wild animals in
game parks were tested for antibodies reactive to E. chaffeensis and E. canis by the
indirect fluorescent antibody assay. Ticks collected from dogs were analyzed by
nested polymerase chain reaction (PCR) to determine Ehrlichia organisms in the ticks.
Overall, 68 (42%) of the dogs had significant antibody titers to E. canis and 61
(38%) had significant titers ( 1/64) against E. chaffeensis. Three wild animal spp.,
which include Ceratotherium simum (white rhinoceros), Antidorcas marsupialis
(springbok) and gemsbok (Oryx gazella) had significant titers (1/64) to E. canis and
none of the animals had significant titers against E. chaffeensis. E. canis DNA could
also be amplified in Haemaphysalis leachi dog ticks.
This study has indicated that dogs and wild animals can act as potential
reservoirs for Ehrlichia, or a closely related organism, in nature.
Severe encephalopathies in children with antibodies reactive with
Rickettsia africae
A-M Pretorius1, R Jacquemard2, E van der Ryst1*, A Venter2, PJ Kelly3
Depts. of 1Medical Microbiology (Virology Division*), 2Pediatrics and Child Health,
Faculty of Health Sciences, UOFS, Bloemfontein, South Africa and 3Biomedical
Research and Training Institute, Harare, Zimbabwe
A number of children with severe encephalopathies presented at the
Universitas and Pelonomi Hospitals in Bloemfontein. No bacterial cause for the
neurologic syndromes in these children could be identified and a viral etiology was
excluded by exhaustive examinations. Rickettsiae was not initially considered as a
possible etiologic factor as there was no history of a tick bite or skin rash. Clinical
examination also revealed no stigmata of tick bite fever, but this could have been
missed as the diagnosis was not specifically considered. Subsequently, sera from
three of the children were tested for antibodies to the spotted fever group (SFG)
rickettsiae using an indirect immunofluorescence assay, as neurologic involvement in
SFG rickettsial infections has been described. All of the children had significant IgM
antibodies against these organisms with titers ranging from 64 to 256. We are
currently trying to amplify rickettsial DNA from the CSF of the fourth child.
Unfortunately, no whole blood from the children was available for PCR assays for
rickettsial DNA. We, therefore, propose that the SFG rickettsiae might be a cause of
severe encephalopathies in children. These infections should be considered in
children with encephalopathies of unknown etiology as the treatment differs from the
standard treatment of encephalopathies in children. Early appropriate treatment with,
for example chloramphenicol, could be life saving.
- 47 -
77B
Detection of antibodies to Ehrlichia sennetsu in human sera from
Malaysia and Myanmar (Burma).
83
Hanson, B., C. Wongsrichanalai, M. Simanowith, J. Antony, B. Belcher, and H.
Paxton.
Integrated Diagnostics, Inc., Baltimore, MD, USA, and Armed Forces Research
Institute of Medical Science, Bangkok, Thailand
Ehrlichia sennetsu (ES), the causative agent of a mononucleosis-like disease, was
first isolated from humans in Japan in the 1950‚s, but little has been learned of its
geographic distribution since then. Likewise, the mode of ES transmission is
unknown, although its antigenic relationship to E. risticii suggests possible
involvement of a non-arthropod vector. During field testing of a dot ELISA multidipstick in Southeast Asia, we detected IFA antibodies to E. sennetsu in human sera
from Malaysia and Myanmar. Among retrospective sera from 54 individuals in two
Myanmar locations and selected on the basis of their multi-dipstick reactivities, 8 had
anti-ES (polyvalent) IFA titers > 1:80. One subject had an IgM titer of 1:640,
suggesting a recent ES exposure. Among sera from 132 Malaysian patients selected
for the presence of antibodies to other rickettsiae and to S.typhi, six evidenced
exposure to ES, with (polyvalent) IFA titers of 1:128 to 1:1024. Two of these also had
significant IgM titers, and a third showed a four-fold rise in titer, implying recent
infections. A 50-sample retrospective serum panel from northwest Thailand had no
anti-ES IFA titers > 1:40. Control IFA tests which suggested no cross reactivity among
E. sennetsu, E. chaffeensis, and HGE supported the proposition that the seroreactivity
reported here was specific for E. sennetsu or a related organism. This indication of
human exposure to ES in Myanmar and Malaysia provides an opportunity to broaden
our understanding of its geographic distribution by larger serosurveys and agent
isolations and to study its mode of transmission and the human immune response to
it. Supported by Dept. of the Army Contract DAMD 17-96-C-6027.
Erythema migrans-like lesions after Dermacentor sp. tick-bite
without evidence of Borrelia burgdorferi infection.
JA. Oteo, JR Blanco, V. Martínez de Artola, P. Anda
Dept. Infectious Disease. H. of La Rioja. Avda. de Viana nº1. 26001 LOGROÑO
(SPAIN).
Erythema migrans (EM) is the best and the most specific marker of Lyme
disease and Borrelia burgdorferi infection which is transmitted by the tick Ixodes
ricinus. We have observed three patients with atypical erythema migrans lesions
(central necrosis) after a tick bite by Dermacentor sp. that showed no evidence of B.
Burgdorferi infection.
Case 1: November 1996. A 67-year-old man developed a rash resembling EM
with central necrosis in right sub-mammalian area 8 days after a D. marginatus tick
bite. A skin biopsy from the EM patient was take and inoculated in C3H/He mice and
into culture (BSK medium) without growth of spirochetes. A PCR (ospA) of the skin
biopsy and the serologic serum samples didn't demonstrate B. burgdorferi or
Rickettsia conorii infection. After 21days of doxycicline the patient was asymptomatic.
A PCR (ospA from a D. marginatus) was negative.
Case 2: March 1995. A 49-year-old woman developed a febrile rash resembling
EM with central necrosis and satellite linfadenopathies 7 days after a tick bite sp.
compatible with Dermacentor sp. in her head. After 21 days of doxycicline the patient
was asymptomatic. The serological studies didn't demonstrated B. burgdorferi or R.
Conorii infection.
Case 3: November 1993. A 59-year-old woman developed a rash resembling
EM with central necrosis in the right arm five days after a Dermacentor sp. tick. The
serological studies didn't demonstrate B. burgdorferi or R. Conorii infection. After 21
days of doxycicline the patient was asympthomatic.
- 48 -
89
Characterization of Bartonella clarridgeiae flagella and
detection of antiflagellin antibodies in patients with
lymphadenopathy.
113
Anna Sander1, Anja Zagrosek1, Karin Oberle1 and Christoph Dehio2
1Institute for Medical Microbiology and Hygiene, University of Freiburg, and 2Max
Planck Institute for Biology, Tübingen, Germany.
Cat-scratch disease (CSD) is caused in the majority of cases by Bartonella henselae. Recently two
case reports indicated B. clarridgeiae as an additional causative agent of CSD. Both species have been
isolated from domestic cats, which are considered as the natural reservoir of these bacteria. Bartonella
species are genetically and phenotypically closely related. However, it was shown that B. henselae may
have pili, whereas B. clarridgeiae possesses multiple unipolar flagellae. The presence of a flagella
represents one of the main differences between B. henselae and B. clarridgeiae.
We purified and characterized flagellae from B. clarridgeiae. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a
polypeptide of 41 kDa. The gene coding for this flagella was sequenced and primers for PCR detection of B.
clarridgeiae DNA have been developed.
Sera from 724 patients with lymphadenopathy, for which CSD was considered in the differential
diagnosis, and 100 sera from healthy controls have been investigated by immunoblot for detection of
antibodies to the B. clarridgeiae flagellin protein. 156 (21%) of these sera had high (512), 229 (32%) had
low (64 to 256) and 339 (47%) had no antibody titers to B. henselae (IFT). Antiflagellin antibodies could be
detected in 28 (3,9%) of the patients sera, but in none of the control sera. No serological cross-reactivity
with other bacteria has been found. Lymph node biopsies from two patients having antiflagellin antibodies
were available. In both of these lymph nodes B. henselae DNA but not B. clarridgeiae flagella DNA could be
detected by PCR methods. However, both patients had additionally high antibody titers to B. henselae.
Our results suggest, that B. clarridgeiae might be a rare, additional cause of CSD. However,
serology alone seems not to be reliable enough for diagnosis of acute B. clarridgeiae infection (as indicated
by the two patients). Further data are needed for determination the role of B. clarridgeiae in CSD.
Serological evidence of Ehrlichiosis in Australia
B. Hudson, V. Lennox*, J. Stenos, and D. Walker.
Department of Microbiology and Infectious Diseases, PaLMS, Royal North Shore
Hospital, St. Leonards, NSW, Australia* , and University of Texas, Medical Branch,
Galveston, USA.
Sera from thirty-seven patients were tested for antibodies to Ehrlichia
chaffeensis, of these, nine patients gave a positive titre of 64 or greater. Eight patients
had a known history for tick bite. The one patient with no known tick bite had an
Ehrlichia titre of 256, was originally diagnosed with leptospirosis (clinically and
serologically) and went on to develop a chronic illness. Five patients had no evidence
of overseas contact with ticks and all had documented tick bites with subsequent
illness. The other two patients had documented tick bites within Australia but because
of their frequent travel to South Africa an overseas exposure could not be ruled out.
Data will be produced showing the clinical syndrome of the nine patients, their
response to treatment and the serological evidence to suggest that Ehrlichia sp. may
be present in Australian ticks and may be a cause of clinical illness post-tick bite.
- 49 -
114
Serologic Evidence of Spotted Fever Group Rickettsia in Novo Cruzeiro Municipality-Minas Gerais State-Brazil.
118
Marcio Galvao; Chequer B. Chamone, Simone B. Calic, Mirtes C. Machado,
Marcia E. A Otoni, Reynaldo Dietze, Cecilia Moron, Hui-Min Feng, Juan P. Olano,
David H. Walker
University of Texas Medical Branch-USA/Universidade Federal de Ouro PretoBrazil/Fundacao Ezequiel Dias-Brazil/DRS Teofilo Otoni-Brazil/Universidade Federal
do Espirito Santo-Brazil.
Brazilian spotted fever is known to occur in the States of Minas Gerais, São Paulo, Rio
de Janeiro, Bahia and Espirito Santo. In 1990 Minas Gerais State initiated a laboratory
surveillance program for Brazilian spotted fever. Between 1993 and 1995, Novo Cruzeiro
Municipality had the highest case-fatality ratio (30%) for Brazilian spotted fever in Minas Gerais
State, and was the municipality selected for a serologic survey for rickettsial diseases. A single
sample of venous blood was drawn from 141 persons among 170 persons living in this region
during the period September 1-5, 1998, three years after the last diagnosed case of Brazilian
spotted fever. Sera were separated by centrifugation and stored at -20o C until tested at the
University of Texas Medical Branch (UTMB)-Galveston-Texas-USA. At UTMB the 141 sera
were screened for the presence of antibodies to Rickettsia rickettsii,Ehrlichia chaffeensis and R.
typhi by the indirect fluorescent antibody (IFA) test at a titer of 1/64. Twenty-six (18%) of the
sera had IFA titers of 1/64 to R. rickettsii. None had IFA antibodies at a titer of 1/64 to R. typhi
or E. chaffeensis. Among the 26 sera that had antibodies to R. rickettsii, 4 had a titer of 1/128, 2
at 1/256, and 1 at 1/512. Nineteen sera among these 26 were tested for the presence of
antibodies to R. rickettsii by Western blot, and only one revealed antibodies to rOmpA and
rOmpB. This evidence demonstrates the importance of investigating further the presence of
another species of Rickettsia in the area and in other regions of Brazil. It will be useful to
attempt to identify other Rickettsia species from vectors (ticks and fleas) by molecular biology
technique, and isolation of rickettsiae from patients, which requires a good surveillance system.
Epidemic Manifestations the Rickettsial Diseases in Ukraine
M. D.Klymchuk
The Ukrainian Centre of the Rickettsiosis Of the Ministry Public Health in Ukraine
Wolhynica (quintana) fever and Marseilles fever may have epidemiological meaning
amongst famous rickettsiosis in Ukraine in contemporare conditions. Although the natural
centres of Q-fever are widely spread all over the territory of the country, Q-fever is manifestated
by separate sporadic diseases, but untimely incomplete diagnostic of the diseases increases
the number of the chronic forms of the infection.
Using high-specific antigen preparations with strains of R.quintana, which are
continuosly cultivated in our laboratory by Weigl’s method, we had determined infection of the
different contigents of population of the agent of disease of Wolhynica fever: antibodies were
defined in 4,23% and differentiated, as Ig G and Ig M. The diseases of people, who infested by
transmitter were diagnosed in retrospection. The characteristic of the activ epidemic process is
defined by the antibodies against R.quintana in children and young people (3,36 - 8,15%) and
amongst certain categories of population, which were infested by the pediculosis too, so the
diagnostic of the infestant is in the transmitter himself
In the south of Ukraine (AR Crimea) in 1996 the epidemic outbreak of Marseilles fever
for the first was studied in before unknown natural centres. The rickettsiosis in sick persons
were characterised by the clinically heavy and middle course (94%). Standardization of the
ternperature was observed when antibiotics of specific action in 4-5 days were used. The two
sick persons with attendant diseases had died. The antibodies against R.conori in the titer 1:101:160 were displayed in family centres of infection in the people, who did not appeal to the
medical help. The wide zone of natural centres with sporadic diseases of people was
determined almost on all the territory of the peninsula, including the steppe zone, although
news about the Marseilles fever in the Crimea was limited mainly by the city of Sevastopol’
during many years. The laboratory diagnostic of diseases was adjusted in endemic zones.
- 50 -
131
First discovery on infection of Spotted Fever Group Rickettsiae
among healthy persons in Guangdong province.
132B
Zhang Jian-zhi 1, Gou Yan 2, He Jin-rong 1, Xu Shi-e 2,Pan Lin-xiang 3
1 Institute of Epidemiology and Microbiology£¬Chinese Academy of Preventive
Medicine£¬Beijing 102206 2 Shantou Medical College, Shantou University, Shantou
515031 3 Meizhou Sanitary and Anti-epidemic Station,Meizhou 514000
Seroepidemiological studies of spotted fever group rickettsiae infection were
performed among 1012 healthy persons in three counties of Guangdong Province by
the method of microimmunofluorescence assay.The results show that the prevalence
of antibodies to R.sibirica were 28.47% ¡¢20.46% and 6.8% respectively in
Dapu,Pingyuan and Mei Counties; that to R.conorii were 27.78% ¡¢13.42% and
25.59% respectively and to R.akari were 21.88%¡¢18.80%¡¢15.49% respectively.
The highest antibody£¨28.47%£© prevalence was found in Dapu County.Prevalence
of antibodies was not correlated with sex¡¢age and profession.The results suggested
that there exsited the natural foci of spotted fever group rickettsiae in Dapu¡¢Mei and
Pingyuan counties of Guangdong province.
Isolation and Identification of HN-98 strain of Spotted Fever Group
Rickettsiae.
Zhang Jian-zhi1,Chang Bin-gong1, Tian Xiaodong, Bi De-zeng1.
1. Department of Rickettsiology, Institute of Epidemiology and Microbiology,Chinese
Academy of Preventive Medicine£¬Beijing, 102206 2 Institute of Military Medicine,
Guang Command,Guangzhou£¬510507
One strain of rickettsiae was isolated from patient with unknown fever from
Qiongzhong county in Hannan Province in 1998 by using embryonated hens eggs and
proved to be the member of rickettsiae by the methods of morphrology, named as HN98 strain after the name of the place and the year which it was isolated. The isolate
was identified by the methods of Microcomplement fixation assay and PCR/RFLP and
compared with known species and strains of SFGR by DNA polymerase chain
reaction and DNA polymerase chain reaction followed by restriction endonuclease
fragment length polymorphism analysis. The results demonstrated that HN-98 strain
was antigenically and genotypically identical to R.sibirica 246.
- 51 -
132C
136A
Chronic Q-fever in a swedish patient
E Franzén-Rohl
Department of Infectious Diseases, Karolinska Hospital,Stockholm, Sweden.
We describe the case of a 42-year old male with a 5 years-long history of
endocarditis, glomerulonephritis, fever, myalgia and anemia. This patient had a preexisting heartvalvular disease with no special treatment. In spring -96 he suddenly
developed, an aortic aneurysm and suspect endocarditis, this lead to acute valvular
replacement and graft in aortic ascendens. All blood cultures were repetitively
negative .As the patient continued with fever and presented a high rheumatoid factor
and hematuria, he was sent to the Department of Rheumatology. (also HLA-B27
positiv.) Nothing specific was found, the biopsy of the kidney only revealed the picture
of Immuncomplex mediated-glomerulonephritis. The diagnose was finally confirmed
by an increase in specific antibody titre against Coxiella burnettii (IgG Phase I higher,
than Phase II) Specific antibodies against Bartonella henselae was also significantly
positive, (low-level) as earlier described ,cross-reactivity occurs in chronic Q-fever
.This is the first Swedish patient , with chronic Q-fever published in the Nordic
countries. This case was not a local outbreak, we assume our patient got his acute Qfever by travelling in Rajastan, India-95. Serology from January-96 shows high
antibody titre against Coxiella burnettii. First he developed an acute Q-fever and then
following a chronic Q-fever disease. Q-fever should not lead to misdiagnosis, provided
serology testing against various agents is performed.
Q-fever endocarditis - First case report from Sweden
Elisabeth Franzén-Röhl1 and Sirkka Vene2,
Department of Infectious Diseases, Karolinska Hospital (1) and Swedish Institute for
Infectious Disease Conbtrol (2), Stokholm, Sweden.
We describe the case of a 42-year-old male with a 5-year history of endocarditis,
glomerulonephritis, fever, myalgia, and anemia.
The patient had a pre-existing non-treated heart-valvular disease when in the spring of
1996, he suddenly developed an aortic aneurysm and suspected endocarditis, which lead to
acute valvular replacement and a graft in the aortic ascendens. The patient remained febrile
and blood cultures were repeatedly negative. As the patient was HLA B27-positive and
exhibited high levels of rheumatoid factor and hematuria, an autoimmune disease was
suspected. A kidney biopsy revealed a picture of immune-complex mediated
glomerulonephritis.
The Q-fever diagnosis was finally confirmed in April 1998 by the demonstration of
Coxiella burnetii phase I and II specific antibodies by a microimmunofluorescence assay (see
below), and antibiotic treatment was initiated.
Sera drawn in 1996 and 1997 were subsequently tested, and the enhanced IgG-titers to
the phase I-antigen, which are considered diagnostic for Q-fever endocarditis, were
demonstrable also in these samples.
As Q-fever is extremely rare in Sweden,we assume that the patient was infected while
travelling abroad (India) in 1995.
Micro-IF titers against C. burnetii phase I and II antigens
Sample dates
Feb. 1996
Nov. 1997
phase I IgM
10.240
1280
IgG
10.240
> 20.480
phase II IgM
> 160
> 160
IgG
10.240
> 20.480
- 52 -
April 1998
320
10.240
160
10.240
Aug. 1998
not tested
5120
not tested
5120
136B
The re-emergence of Siberian tick typhus: field and experimental
observations.
1
1
1
137
1
N.V. Rudakov* , I.E. Samoilenko , V.V. Yakimenko , T.A. Reshetnikova , S.N.
Chpynov1, D.H. Walker2 .
1Omsk Reseach Institute of Natural Foci Infections, Russia - 2University of Texas
Medical Branch, Galveston, TX, USA
Epidemiologically active foci of Siberian tick typhus (STT) are wide spread in
Asiatic part of Russia and North Kazachstan. The main vectors of Rickettsia sibirica
are Dermacentor (Subgenus Serdjukovia) ticks. Field and experimental observations
during the last 20 years showed no greater proportion of ticks containing SFG
rickettsiae. New approach to quantitative and qualitative characterization of SFG
rickettsiae in STTís foci was used. Apathogenic SFG rickettsiae were detected in
epidemiologically active foci of STT and STT-free territories. These agents deffered
from R.sibirica in virulence, antigenic characteristics with monoclonals, transstadial
and transovarial transmission and were ìnoncultivatedî. Differences in results of SFG
screening of ticks from endemic and nonendemic territories were showed in indirect
hemagglutination, ELISA and direct immunofluorencent assay with polyclonal SFGantibodies. Tick experimental model was used as a new tool for study of noncultivated
SFG rickettsiae. Successful transovarial and transstadial transmission of rickettsiae
from STT foci was detected in 4 generations of ticks (P-F1-F2-F3-F4) within 3,5 years
(time of examination). Possibility of interference between apathogens (Kazachstan
SFG strains) and virulent R.sibirica (Altay strain) was detected. New data maybe used
for understanding of re-emergence of STT foci.
Seroepidemiological survey of Bartonella henselae infection in
Catalonia, Spain
I. Sanfeliu, N. Cardeñosa, F. Segura, G. Diestre, T. Muñoz and B. Font
Corporació Sanitària Parc Taulí. Sabadell. Barcelona. Spain
INTRODUCTION: Bartonella henselae is the etiological agent of cat-scratch
disease. Seroprevalence studies in normal population are necessary to get a better
understanding of this disease. The aim of this study was to know the prevalence of
infection against Bartonella henselae in healthy individuals sera in our area
(Catalonia).
MATERIALS AND METHODS: Between September 1993 and January 1994,
219 sera were collected in Hospital de Sabadell. The study population was stratified
by age, sex and demographic area. All serum samples were tested by indirect
immunofluorescence assay using a commercially available antigen (Bartonella IFA
IgG Substrate Slide, MRL DIAGNOSTICS, USA). All sera with a titre  1:64 were
considered positive.
RESULTS: Of the 219 sera studied, 19 had antibodies to B. henselae. Ten sera
were from women and 9 from men. The age range for seropositive subjects ranged
from 3 to 76 years old. The overall seroprevalence was 8.7%. When these results
were compared with previous serological studies carried out with the same sera, we
found two which also had antibodies against R. conorii antigens detected at similar
titers by IFA; another one also reacted against C. burnetii antigens detected by IFA;
and three other ones were also positive against B. burgdorferi antigens detected by
EIA.
CONCLUSIONS: These results corroborate the presence of B. henselae
infection in Catalonia.
- 53 -
166
Antibodies against Coxiella burnetii in patients with valvular heart
disease residents in an endemic area of Q fever
a
.b
178
b
Pascual-Velasco F ., Montes M , Cilla G .
a Department of Internal Medicine, Hospital Comarcal de Laredo, Cantabria, Spain. b
Microbiology Service, Laboratorio Unificado de Donostia, Gipuzkoa, Spain. E-mail:
ludserolo@chdo.osakidetza.net
To determine the presence of subclinic chronic Q fever in an endemic area of Q
fever (Eastern Cantabria, Spain)(1) among subjects with valvular heart disease living
in this area.
Between November 1994 and March 1996, 51 subjects were serologically
studied for Q fever infection. All of them suffered from a diversity of valvular heart
diseases confirmed by Eco-Doppler and with different grades of heart failure but
without clinical data of endocarditis. Fifty percent of the cases were degenerative
aortic stenosis, 24% aortic and/or mitral valve prostheses, and the rest mitral-aortic
and/or tricuspid failure of diverse etilogy. By sex, 22 were males and 29 females. The
average age of the patients was 76 years old (54-96 years). C. burnetii IgG antibodies
were determined by IFI using C. burnetii antigen in phase II. In the positive samples,
C. burnetii IgG antibodies in phase I were determined. The methodology used has
been described elsewhere (1).Twenty out of 51 patients (39,2%) had IgG antibodies
against C. burnetii in phase II (1/512 the highest title). Only two of them had IgG in
phase I, being 1/128 the highest title. No one had serological criteria for chronic Q
fever. Comparing these results with those obtained in the general population in
Eastern Cantabria (subjects=595; prevalence of IgG phase II= 48,6%)(1) there were
not statistical significative differences.
Although Eastern Cantabria is an endemic area of Q fever, we have not
detected, during the study period, any suggestive case of chronic Q fever in a group
at risk for it.
(1) Pascual-Velasco F, Montes M, Marimón JM, Cilla G. High seroprevalence of
Coxiella burnetii infection in Eastern Cantabria (Spain). Int J Epidemiol 1998; 27: 142-145.
Lice and lice-borne rickettsioses in Ukraine
I. Kurhanova, M. Klymchuk, S. Lubinski, M. Kitzara, Z. Kos, N. Basarab
Lviv Research Institute of Epidemiology and Hygiene, Lviv, Ukraine
Head lice infestation prevails in current situation in Ukraine, occuring mainly
among schoolchildren. Poor density predominates (less that 10 lice or nits); only dry
nits are discovered in 50% of all cases; the hotbeds with one infested man form 95%.
The number of mixed and body lice infestation with high rate (50-100 body and head
lice) increases among homeless, mentally sick, old people.
Clinical findings of epidemic typhus and rise of antibodies against R. prowazekii
among children, young-, middle-aged people are absent. The cases of Brill-Zinsser
disease are observed among people over 50. The antibodies against R. prowazekii in
CFR (titre 1:10-1:20) are discovered among 2,82% of these people. In indirect
immunofluorescent test the antibodies are presented as IgG.
The diseases with fever are manifested among people of different age. In these
cases the rise of CF-antibodies (titre 1:40-1:80> against B. quintana with IgG or/and
IgM is discovered in indirect immunofluorescent test. Seroprevalence in the human
population of the abovedescribed age group comprises 5,22%.
During last 15 years the seroprevalence antibodies in CFR against R.
prowazekii reduced from 9,58% to 0,69% (t>2) and against B. quintana - from 7,85%
to 5,41% (t>2).
Head and body lice from different regions of Ukraine were infected with B.
quintana, but not with R. prowazekii.
Our data show absence of R. prowazekii circulation and presence of B.
quintana circulation due to infected vectors, the diseases among people and
seroprevalence among population.
- 54 -
179
On possible effect of R. sibirica ecology on Asian Rickettsiosis
Anatoly S. Obert
The Altai State Medical University, Institute for Water Environmental Problems,
Siberian Branch of the Russian Academy of Sciences
The Altai Territory is one of the asian rickettsiosis powerful foci in West Siberia.
At present the growth of asian rickettsiosis cases in observed and in the process the
number of seronegative people in beeing increased. The last years investigations
(N.V. Rudakov, 1998) point out the fact that exept R. sibirica, other species of
rickettsiosis circulate, which are found in the same disease carriers. This may effect
the disease course. In this connection the aim of investigation is to compare the
disease course of patients with different immune response in typical serologic
reactions (CFT and IHT) with pathogene antigen.
Two groups of patiens with asian rickettsiosis: seropositive (31 persons) and
seronegative (42 persons) were formed by accidental selection. About 27 clinical,
laboratory and epidemiological indexes were taken as the comparison criteria. Places
of residence, the age of patients as well as methods of examination and treatment
agreed. The results obtained were statisticaly processed. The illness was taking its
normal course for the patients from both groups. This gave ground to asian
rickettsiosis diagnosis without regard to serologic examination results. More careful
analysis allowed to reveal specific differences in average duration of incubative
period, frequency of some intoxication characters and initial complex in the place of
infection atrium as well as in indexes of peripheric blood and results on serology test
of blood serum (activity of aspartate- and alanine-aminotransferase). One can't
exclude the possibility of differences occurrence between some strains of Rickettsia
sibirica in virulence and immunogenicity, homogeneity and genotype (N.V. Rudakov,
1998) that may influence the response of infected human organism.
- 55 -
185
Tuesday June 15th
Slide session 3
Emerging Rickettsioses
Tick-borne lymphadenopathy (TIBOLA) a Rickettsia slovaca
infection?
Lakos, András* and Raoult, Didier**
*Center for Tick-borne Diseases, Budapest, Hungary, **Institute for Rickettsioses,
Medical Department, WHO Collaborative Center for Rickettsial Reference and
Research, Marseille, France
Three children with similar symptoms were observed at the Center for Tickborne Diseases, Budapest in the years 1997 and 1998. All three patients had been
bitten by a "very large" tick on the occipital scalp region. A few days after the tick bite,
a huge lymphadenopathy developed around the tick bite on the occipital scalp region
and in the neck behind the sternocleidomastoideal muscle. A necrotizing papula
(eschar) developed at the site of the tick bite in every patient. A prominent discharge
was seen from this eschar in one case. The eruption was surrounded by a circular
erythema in two cases. All three patients healed, except alopecia remained at the site
of the eschar in two cases. Rickettsia slovaca infection was proved in one child by a
seroconversion in IgG and IgM antibody during reconvalescence. R. slovaca infection
was proved in another girl with elevated IgG and IgM antibody titer and suspected in a
boy with elevated IgG antibody only. Antibodies for Bartonella henselae, B. quintana,
Francisella tularensis and Borrelia burgdorferi as well as human granulocytic
ehrlichiosis (HGE) agent were also tested but all these were negative.
- 56 -
108
Rickettsia felis: the etiologic agent of a case of rickettsiosis in the
Yucatan
1
1
1
1
147A
1
Zavala-Velasquez JE, Ruiz-Sosa JA, Jimenez B, Vado-Solis I, Zavala-Castro
J, 2Walker DH.
1Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico, and 2Department of
Pathology, University of Texas Medical Branch, Galveston, Texas, US
In 1993, a rickettsiosis of the SFG not recognized previously and clinically masquerading as dengue
fever was documented in Mexico. Forty percent of patients had IgM titers to Rickettsia rickettsii and/or R.
akari equal or greater than 1:128. The clinical signs and symptoms included fever (100%), myalgia (95%),
headache (85%), and rash (85%). Later studies showed that the seroprevalence of rickettsial antibodies in
the population of the State of Yucatan was 5%, with a seroreactivity to the LPS bands of R. akari. The
clinical diagnosis is difficult; however, this is the first definite report of a rickettsial illness diagnosed in a 34
year old indigenous Indian female, that lives in the rural eastern region of the Yucatan Peninsula
(Valladolid). She lives in close contact with dogs, cats and several other animals including chickens and
turkeys. The patient gave a history of approximately 15 days of illness prior to admission; she had fever,
neck and head pain, myalgia, focal hemorrhagic conjunctivitis, hearing loss, signs of meningeal irritation,
epidermal lesions described as furuncules that evolved to ulcers on the arms and chest and Babinski
reflexes. On admission the lab tests showed anemia (Hgb 7 gm/dl) and leukocytosis (19,000/l), with 15
lymphocytes and 81 segmented PMNs. A skin punch biopsy was performed on one of the lesions of the
upper chest that had slightly raised borders, a bluish periphery and central crust that measured about 1 cm.
She was discharged with improvement of the signs and symptoms a week after admission and treatment
with antibiotics (amikacin, chloramphenicol and doxycycline). Microscopically the biopsy showed an
inflammatory process with mononuclear cells, and no microorganisms were visualized with the stains
performed in Mexico. The IFA in acute stage was negative, and in convalescent stage (6 weeks later)
presented titers of 1:64 for R. rickettsii, R. akari, and R. typhi. PCR of whole blood DNA using primers for
the 17-kDa antigen gene was negative; however, the biopsy was positive. The 426 bp amplified product
was cloned and sequenced. The sequence represents the nucleotide positions 91 to 516 of the complete
17-kDa genus-common antigen gene of R. rickettsii. A 100% homology with R. felis was observed for the
240 internal sequence of the nested product. It also had the following homologies: R. rickettsii (93.9%), R.
conorii (93.7%), R. typhi (87.8%), and R. prowazekii (87.1%). Two sites for the Alul restriction enzyme were
recognized in the 127 and 236 positions of the sequence, similar to R. felis, R. rickettsii and R. conorii. We
believe that the etiologic agent in this case is R. felis, the second report ever of a human clinical case for
this species, the first associated with an eschar, and the first in the tropics.
Etiology of febrile illnesses after a tick bite in Slovenian patients
with leukopenia and/or thrombocytopenia
S. Lotric-Furlan, M. Petrovec, T. Avsic-Zupanc, William L. Nicholson, John W.
Sumner, James E. Childs, F. Strle.
University Medical Centre and Institute of Microbiology and Immunology, Medical
Faculty Ljubljana, Slovenia, Viral and Rickettsial Zoonoses Branch, Centers for
Disease Control and Prevention, Atlanta, GA, USA
A prospective study on the etiology of febrile illnesses occurring within six
weeks after a tick bite in adult patients was conducted at our department from 1995 to
1997. Patients were tested for the presence of serum antibodies to tick-borne
encephalitis (TBE) virus, Borrelia burgdorferi sensu lato, Rickettsia conorii, Ehrlichia
chaffeensis and human granulocytic ehrlichia (HGE) agent, as well as by polymerase
chain reaction (PCR) for the presence of the HGE agent and E. chaffeensis.
Demonstration of IgM antibodies against TBE virus by ELISA test was interpreted as
an indicator of a recent infection. Serum antibody titres ? 1:128 to HGE agent and E.
chaffeensis, titres ? 1:80 to R. conorii, and serum titres ? 1:256 to B. burgdorferi were
interpreted as positive by IFA. A subset of 42 patients with leukopenia and/or
thrombocytopenia found at first visit is presented. Positive serum antibody titres
against at least one of the tick-borne agents were found in 21(50%) patients. IgM
antibodies to TBE virus were detected in 12/42 (28.6%). Positive serum antibodies
titres to B. burgdorferi were found in 6/42 (14.3%) patients; in two of them erythema
migrans was present and B. burgdorferi isolated from skin. No antibodies to R. conorii
were established. Three of 42 (7.1%) patients fulfilled criteria for acute HGE by
positive PCR results and seroconversion. In two of them antibodies to E. chaffeensis
were also detected. Thus, the most common known cause was the infection with TBE
virus followed by B. burgdorferi and HGE agent.
- 57 -
87
Granulocytic Ehrlichiae infections in humans, ticks and wild
mammals in western Switzerland
78
Jorge S. Liz, Laurence Anderes, Kurt Pfister, Lise Gern, Bernard Rutti and
Michel Brossard
Institute of Zoology, University of Neuchâtel, 2007 Neuchâtel, Switzerland
Granulocytic Ehrlichiae infection is a common disease in cattle in some areas of Switzerland where
the infection is caused by Ehrlichia phagocytophila and transmitted by Ixodes ricinus ticks. E.
phagocytophila is closely related to the causative agent of the human granulocytic ehrlichiosis (HGE)
described in the United States and recently discovered in Europe. E. phagocytophila, HGE agent and E.
equi are members of the E. phagocytophila genogroup, commonly named granulocytic Ehrlichiae.
We examined 275 Swiss human sera for IgG antibodies to granulocytic Ehrlichiae by
immunofluorescent assay, using E. phagocytophila-infected bovine neutrophils (Swiss strain) and HL-60
cells infected by HGE agent (U.S. strain) as antigens. Fifty (18.2 %) were reactive to both antigens (titer 
1/128). Four seroconversions were observed. All the sera came from patients who had sustained tick bites
prior to the onset of their illness and were suspected of Lyme borreliosis. Previous observations showed
that 84 (30.5 %) had antibodies to Borrelia burgdorferi (IgM and/or IgG). Among the granulocytic Ehrlichiae
positive sera, 15 reacted to B. burgdorferi (IgM and/or IgG) as well.
Presence of granulocytic Ehrlichiae in I. ricinus ticks and wild rodents was demonstrated. We used
a specific PCR-based test to study a total of 346 unfed ticks (nymphs and adults) collected by flagging
vegetation and 43 batches of ticks (larvae and nymphs) taken from 43 trapped rodents (Apodemus
sylvaticus, A. flavicollis and Clethrionomys glareolus). Respectively, six host-seeking ticks (three females
and three nymphs) and four batches of larvae (collected on four C. glareolus voles) were found to contain
ehrlichial DNA. Granulocytic Ehrlichiae DNA was also detected in spleen, liver and ear samples from the
rodents infested by positive ticks. Serology in large wild mammals showed that alpine chamois (Rupicapra
rupicapra) and roe deer (Capreolus capreolus) have antibodies against E. phagocytophila.
In this study, we identified C. glareolus vole as one of the reservoir host of granulocytic Ehrlichiae
which is responsible for transmission of the infectious agent to ticks and demonstrated that Swiss
population is in contact with granulocytic Ehrlichiae infected ticks
Limits To Infection With The Human Granulocytic Ehrlichiosis
Agent
Thomas N. Mather1, Michael J. Mauel1, Stacey J. Carlton1, Jennifer R. Douglas1,
Nathan J. Miller1, Kirby C. Stafford2 and Robert F. Massung3
1University of Rhode Island, Kingston, RI, USA, 2Connecticut Agriculture Experiment
Station, New Haven, CT, USA 3Centers for Disease Control & Prevention, Atlanta,
GA, USA
In the USA, disease caused by the human granulocytic ehrlichiosis (HGE)
agent generally has emerged with epidemiologic similarity to other Ixodes scapularisassociated infections, including Lyme disease and human babesiosis. Although
differences within the tick-host-pathogen paradigm, including broader vector and host
competence and shorter transmission delays following tick attachement, provide
opportunities for higher levels of HGE risk. However, in Rhode Island HGE infection
appears to be lower than Lyme and babesiosis. To date, four 16S rDNA variants to
the HGE agent have been detected infecting wild hosts and ticks from Rhode Island
but not from adjacent Connecticut. Sequence results of PCR products from over 75
Rhode Island I. scapularis revealed the presence of the HGE agent and 2 GE
variants: 12% of ticks contained 16S rDNA sequences typical of the HGE agent and
9% contained sequences typical of a variant found previously in deer and ticks. Most
GE-infected ticks (79%) in Rhode Island were infected with a yet unreported variant
differing from the HGE agent by 2-base pairs on the 16S gene. This variant also was
recovered from mice and chipmunks. In contrast, all 16S rDNA sequence results from
PCR positive Connecticut
I. scapularis (n=25) were similar to the HGE agent.
- 58 -
93
Temporal and spatial dynamics of Ehrlichia phagocytophila
transmission in northeastern USA.
Durland Fish and Michael Levin, Franka desVignes, Joseph Piesman
Dept. of Epidemiology and Public Health, Yale School of Medicine, 20 College St.
P.O. Box 208034, New Haven, CT 06520 USA - Bureau of Communicable Disease
Control, Massachusetts Dept. of Health, 305 South St. Rm 506F, Jamaica Plains, MA
02130 USA - Centers for Disease Control, Div. Vector-Borne Infectious Diseases,
P.O. Box 2087, Fort Collins, CO 80522 USA
The distribution of Ehrlichia phagocytophila, the presumed agent of human
granulocytic ehrlichiosis in the U.S., is unstable in both time and space. Infection
prevalence in the nymphal stage of the vector tick Ixodes scapularis varies
dramatically among generations and between habitats. These observations are in
sharp contrast to Borrelia burgdorferi, which is remarkably stable throughout time and
space in this region, and transmitted by the same tick species. Differences in reservoir
host species composition may be a likely explanation. Transmission by infected
nymphal I. scapularis is equally as efficient with E. phagocytophila as with B.
burgdorferi (80%) and both pathogens are transmitted simultaneously by co-infected
ticks. However, E. phagocytophila was observed to be transmitted from individual
ticks to individual rodent hosts within 24 hrs, compared to 48 hrs for B. burgdorferi.
These observations have important implications in the natural maintenance cycle of E.
phagocytophila and in the epidemiology of human granulocytic ehrlichiosis in the U.S.
- 59 -
120
Tuesday June 15th
Poster session 4
Diagnosis and Treatment of Rickettsioses
Seroepidemiological survey of Rickettsia typhi infection in
Catalonia, Spain
N. Cardeñosa, I. Sanfeliu, F. Segura, G. Diestre, T. Muñoz and B. Font.
Corporació Sanitària Parc Taulí. Sabadell. Barcelona. Spain
INTRODUCTION: Murine typhus seems to be endemic in the South of Spain
where clinical cases have been reported. Not very much is known about this disease
in our country (the causative agent has never been isolated, reservoires and vectors
have hardly been investigated, and only fragmentary seroprevalence studies are
available). In this study we attempt to evaluate the prevalence of antibodies against R.
typhi in human beings in our region (Vallès Occidental, Catalonia, Spain) in order to
confirm the existence of this infection in Catalonia.
MATERIALS AND METHODS: Between September 1993 and January 1994,
219 sera were collected in Hospital de Sabadell. The study population was stratified
by age, sex and demographic area. All serum samples were tested by indirect
immunofluorescence assay using a commercially available antigen (R. typhi IFA, MRL
DIAGNOSTICS, USA). All sera with a titre  1:40 were considered positive.
RESULTS: Of the 219 sera studied, 19 had antibodies to R. typhi. Seven sera
were from women and 12 from men The age range for seropositive subjects ranged
from 9 months to 85 years old. The overall seroprevalence was 8.7%. Ten (52.6%) of
the 19 sera with positive serological test had titers > 1:40. When these results were
compare d with previous serological studies carried out with the same sera, we found
two which also had antibodies against R. conorii antigens detected at the same titers
by IFA; another one also reacted at the same titer against C. burnetii antigens
detected by IFA; and two other ones were also positive against B. burgdorferi
antigens detected by EIA.
CONCLUSIONS: These results corroborate the presence of R. typhi infection in
Catalonia.
- 60 -
1
Ticks species that infest to human in a rural area of Spain.
F.J. Merino1, J.L. Serrano2, A. Encinas3, T. Nebreda1, A. Campos1.
1Servicio de Microbiologia, Hospital General, Soria, 2Servicio de Sanidad y Bienestar
Social, Soria, 3Facultad de Farmacia, Salamanca; Spain.
11A
Ticks are arthropods of great importance as vectors for the transmission of
infectious diseases to humans. In order to determine what species infest humans in
our region, in years 1997 and 1998, all the ticks from the patients who went to the
Centres of Health of our province presenting a tick bite were examined.
One hundred and two ticks were identified. The distribution by species was 62
of Dermacentor marginatus, of which 33 were females and 29 males; 16 Ixodes
ricinus, of them 8 were nymphs, 4 larvae and 4 females; 8 Haemaphysalis punctata, 6
females and 2 males; 6 Rhipicephalus bursa, 4 females and 2 males; 7 Hyalomma
marginatum, 4 females and 3 males; 2 Argas reflexus, with a male and a female and a
male of Dermacentor reticulatus.
In relation with the seasonal distribution, D. marginatus predominates in autumn
(32 cases), I. ricinus specially in spring (10 cases) and autumn (5 cases), H. punctata
predominates in autumn (6 cases), H. marginatum in spring (5 cases) and R. bursa in
summer (5 cases).
The most important conclusion of this work is the predominance of D.
marginatus over the rest of the species, representing the 60% of all the ticks found.
Also relevant is the absence of Rhipicephalus sanguineus, considered the main vector
of the boutonneuse fever, in spite of the fact that our area is an endemic zone for this
disease.
Epidemiological and clinical features of Mediterranean Spotted
Fever in central Tunisia
M. Chakroun*, A. Letaïef**, F. Ben Romdhane*, N. Kaabia**, H. Trabelsi**, N.
Bouzouaia*, L. Jemni**
*CHU F. Bourguiba Monastir, **CHU F. Hached, Sousse ; Tunisia
Mediterranean spotted fever (MSF) is still endemic in Tunisia, where it was first
described in the beginning of this Century. We reviewed in this report 179 cases
(126M, 53F) of MSF diagnosed from 1984 to 1998. Diagnosis was based on
epidemiological, clinical features (Raoult score) and confirmed by serology (IFI)
among 120 patients (67%). Most patients were from rural or suburban areas; 167
(94%) of patients were observed from July to October and 123 had dogs at proximity.
Fever and rash were present among 177 patients (99%) each. «Tache noire»
was observed in 135 cases (77%). Extra-cutaneous manifestations were especially
meningism (4 cases), pulmonary manifestations (4 cases) and pericarditis (1 case).
Biological findings were characterized by: leucopenia or normal WBC in 133
cases (75%), thrombopenia in 34 (28%), elevated transaminases in 62 (46%), and
hyponatremia in 39 cases (28%).
Treatment was based on cyclines in 99 cases and fluoroquinolones in 73
cases. 7 patients were untreated. Ail patients recovered except one death resulted on
severe MSF.
- 61 -
27B
An epidemiological study of tsutsugamushi disease in Shanxi, P.
R. China and the gene analysis of the isolated Orientia
tsutsugamushi strains.
40B
Chen Xiangrui Yu Qiang Zhang Yongguo Niu Hua Zhang Xueying Cheng Cong.
Institute of Microbiology & Epidemiology Beijing P. R. China 100071
Right after the first case of Tsutsugamushi disease was reported in Shanxi
province, P. R. China in 1995, a thorough epidemiological study on this disease had
been carried out in this area. The three-year investigation revealed that, the affected
area had extended to contain three counties; Leptotrombidium apodemi was believed
to be the dominant mite species with a percentage of 66.1% among the collected
mites, and Cricetuls triton was the dominant rodents in this area; while the prevalence
of antibodies to O. Tsutsugamushi was 8.7% among people.
Four strains were isolated from three patients(Sxh951, 952, 953) and one
Cricetuls triton(Sxm97) respectively. LD 50 of these strains to mice varied from 5.5 to
8.0. A pair of primers derived from 56 kDa protein gene were used to amplify this
gene from three isolated strains(Sxh951, 952, 953) and 7 references. RFLP analysis
suggested that the three isolated strains belonged to the same genotype with some
difference from the reference strains. Meanwhile, 56kDa protein gene of Sxh1 was
sequenced, and the resulting sequence was analyzed together with 12 reference
strains using CLUSTALW program and PHYLIP software package. The result
revealed that Sxh951 and Yonchon shared highly similarity in the 56 kDa protein gene
sequence.
Prevalence of antibodies to Rickettsia typhi in Southern Croatia
Volga Punda-Polic1, Sanda Sardelic1, Nikola Bradaric1, Zorana Klismanic-Nuber2
1Medical School Split and University Hospital Split; 2Institute for Public Health, Split,
Croatia
A seroepidemiologic survey was undertaken in the middle part of Southern
Croatia (eastern coast of Adriatic Sea) where the human cases of murine typhus have
been sporadically observed. The prevalence of antibodies reactive with Rickettsia
typhi was investigated by indirect immunofluorescence assay (IFA). Of the 565 human
sera analyzed, 121 (21.4%) showed significant antibody titers (1:80 or higher).
Antibodies to R. typhi were detected more frequently among persons who had contact
with animals. No significant gender-dependence could be proved, in spite of an
apparent higher seropositivity in female sera (22.2% vs.19.5%). The results show that
inhabitants in the area are clearly being exposed to typhus group rickettsiae.
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45
Epidemiology and vaccineprophylaxis of coxiellosis in Russia
Fatalieva S.F., Fetisova N.F.
Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia
51
Coxiellosis in Russia, since its official registration (1957), is characterised by
increasing of morbidity average each five years as well by increasing of quantity of
cases since 1992. Facts of intensive epidemic manifestation was registered in the
following regiones: Central Chernozem, North-West, Povolgie, West-Sibiria.
Coxiellosis was mostly registered in the Astrakhan Region ñ 17,3 on 100 000 of
population. More than 100 outbreaks of coxiellosis took place in each of Vologda,
Astrakhan, Novosibirsk and the Altay Kray. Interheard foci of coxiellosis developed
and lasted for a long time. There we observed also chronical forms of coxiellosis on
humans as a endocarditis, hepatitis, lesion of lungs. Proportion of ill townspeople,
especially on endemic territories, increased and 80% of cases fell on persons at the
age of 20 to 50. Epidemiology of coxiellosis needs improvement of
vaccineprophylaxis. The inactivated combined vaccine (ICV) from C.burnetii phase I ,
strain ìLuga ñ 1î against coxiellosis was worked out. The study of protective properties
of ICV on guinea pigs showed a sufficient immunological efficacy and seroconversion
in 100% of the cases. The same results we had in trials on volunteers with serological
conversion in 55% of the cases by IFT and in 85% of the cases by IFA. We got a
patent ( Nr. 2094057 from 27.12.1997, ìThe method for preparation of vaccine against
Q feverî by Tarassevich I., Fatalieva S., Yablonskaya V. et al.).
Detection of Spotted Fever Group Rickettsia dna in patient’s blood
samples by polymerase chain reaction method
Wu Yimin Liu Xinxin Hulingmei Wei Anming Zhang Zhiqiang Yang Qing
Institute of Medical Science,PLA,Shenyang,P.R.China.
In the north of China, spotted fever group rickettsia(SFGR) exist extensively.
The diagnosis of spotted fever(SF) was mainly depended upon the serologic methods.
Because it is very difficulty to find and diagnosis SF early. The development PCR
technology provided a new diagnosis method for SFGR.In this paper, we used a pair
of primers, which were derived from 190Kda R.rickettsii antigen gene, to amplify
SFGR DNA in patient’s blood specimen directly. 7 persons, who were from the
Suifenhe and Dongning area in Heilongjiang province in China, were bited by ticks
and shown SF clinical symptoms, such as fever, lymphoid node swollen and rash.
Their blood specimens were detected by PCR. The results show that when these
samples were firstly amplified, there were not any DNA bands in the agarose
electrophoresis, but when the first PCR products were amplified again, there was a
obvious band in the sample H-5, which molecular weight was 532bp and identical with
the positive control. Further RFLP anlysis with Pst I indicated that H-5 strain was
identical with R.Heilongjiang 54 strain, but there were significant difference between
H-5 and international reference strain, R.sibirica 246 strain, R. conorri simko strain,
R.rickettsii R strain. Our results suggested PCR is a rapid, simple and sensitive
method, which could be used.
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54B
Surveillance of Reported Cases of Rocky Mountain Spotted
Fever in the United States: 1993 - 1996
95
Treadwell TA, Clarke MJ, Holman RC, Krebs JK, Paddock CD , Childs JE
Between 1993 and 1996, 2,313 cases of Rocky Mountain spotted fever (RMSF)
were reported to the Centers for Disease Control and Prevention (CDC) by 42
states and the District of Columbia through the National Electronic
Telecommunications System for Surveillance (NETSS). Case report forms (CRFs)
were submitted for 1,659 persons of which 1,168 (70%) were confirmed cases. The
South Atlantic region accounted for the largest percentage of reported cases (52%)
followed by the East South Central (14%) and West South Central (13%) regions.
The average annual RMSF incidence rate for this time was 2.3 cases per million
persons. The annual incidence rate of RMSF rose from 1.8 in 1993 to 3.3 per
million persons in 1996 based on NETSS. The average rate was highest (2.8)
among children 5-9 years of age and lowest (0.9) among adults 20-29 years of age.
The case -fatality rate was highest among persons older than 70 years of age (9.0
per 100 cases) and lowest among children less than five years of age (1.7 per 100
cases).
Incidence of Q fever in Gipuzkoa (Basque Country, Spain) from
1984-november 1998
Montes M., Cilla G., Iturzaeta A., Iglesias L., Pérez-Trallero E.
Microbiology Service, Laboratorio Unificado de Donostia, Gipuzkoa, Spain. E-mail:
ludserolo@chdo.osakidetza.net
Q fever is a worlwide zoonosis which has a broad spectrum of clinical
manifestations. Gipuzkoa had the highest incidence of Q fever in Spain. The aim of
this study was to inform about the epidemiological data of Q fever in Gipuzkoa.
Material and Methods. The province of Gipuzkoa is 1997 km2 with a population
of 676,208 inhabitants. It bordered Cantabric sea and France to the North. C. burnetii
IgG and IgM antibodies were assayed by IFI (antigen in phase II from BioMerieux,
France) using anti-human IgG (goat) marked with fluorescein conjugate. All diagnosed
cases had seroconversion, and/or IgM 1/256 after absortion with anti-IgG (RF
Absorbens, Boehringwerke, Munich, Germany).
Results. Among patients with compatible clinic manifestations and the above
criteria, 979 cases of Q fever were diagnosed in the 15 year period, being the annual
rate to the period 1984- November 1998 of 9.65 cases/105 inhabitants. 75.6% of the
cases were young adults, 75.4% occurred in males. The seasonal distribution of the
cases showed that 772 cases (78.9%) appeared in the first six months of the year.
The most frequent disease was pneumonia. Endocarditis was detected in only 2
patients. No mortality was attributed to the infection.
Conclusions. 1.- The incidence of Q fever in Gipuzkoa is very high. 2.- Most of
the Q fever cases occurred in young adult men and during the spring time. 3.- Q fever
causes pneumonia and febrile illness and no complications were derived of them. 4.Endocarditis was infrequent in this high endemic area.
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106
Mediterranean Spotted Fever in the elderly
Feliu Bella, Elena Espejo, Dolors Armengol, Adolfo Rey, Marta Mauri, Rosa
Espejo
Hospital de Terrassa. Terrassa, Spain.
124
OBJECTIVE: To study the characteristics of Mediterranean spotted fever (MSF)
in the elderly.
METHODS: Prospective study of patients aged over 65 with MSF confirmed by
indirect immunofluorescence.
RESULTS: 49 consecutive patients (55% males) were studied. The mean age
was 74.8  6.5 yr (range: 66-95). Underlying conditions other than advanced age were
present in 35 patients (71%). The main clinical manifestations were fever, tache noire,
rash, headache, and arthromyalgia. Gastrointestinal symptoms occurred in 20 cases
(41%). Thirteen patients (27%) had severe forms of MSF, a figure significantly higher
than in younger patients (7%; P= .0001). The main manifestations of severity were:
consciousness impairment (16%), hypotension (12%), acute renal failure (10%),
edema (10%), pneumonitis (8%), hypoalbuminemia (18%), hyponatremia (8%),
hypoxia (4%), and rhabdomyolysis (6%). Severe forms were more frequent in patient
with underlying conditions (31% vs. 14%). Thirty-five patients (including 9 cases with
severe MSF) received one-day doxycycline therapy (two oral doses of 200 mg
separated by a 12-h interval) and the other patients received miscellaneous
therapeutic regimens. In the first group of patients, the fever disappeared after 2.6 
1.1 days and the remaining symptoms (headache, arthromyalgia) disappeared after
3.7  1.3 days of therapy. Outcome was favourable in all cases, but asthenia
persisted for several weeks in most patients.
CONCLUSIONS: Severe forms of MSF are more frequent in the elderly. Oneday doxycycline therapy is very effective in these patients.
Seroprevalence of coxiellosis in cattle, sheep and humans in the
east of Turkey
B. Cetinkaya, H. Kalender, H.B. Ertas, A. Muz, N. Arslan, H. Ongor and M. Grcay
Serum samples collected randomly from 416 cattle in 48 herds and 411 sheep in 47
flocks in 8 differenr locations between June and November 1998 were examined by indirect
fluorescent antibody test (IFA) to determine the prevalence of Q fever in cattle and sheep
populations in the east of Turkey. Age, sex, breed, tick control and abortion history of the herds
and flocks were also recorded. In addition, 102 serum samples were collected from apparently
healthy humans that are at risk of contracting disease such as farmers, veterinarians, abattoir
and laboratory workers and veterinary students. Coxiella burnetii Spot IF kits manufactured by
Bio-Merieux were used to examine serum samples. The test procedure was carried out
according to the manufacturer's instructions. In the examination of sera collected from cattle,
seropositivity was determined in 5.8% (24/216) of the animals in 17 (35.4%) herds, overall. The
prevalence of disease was 4.6% (7/152) in males and 6.4% (17/264) in female cattle. The
seroprevalence ranged from 3.0% to 7.7% in different locations. It was 4.4% (11/251) in cattle
between 0-2 years of age and 7.9% (13/15) in cattle older than 2 years. However, the
differences by agen sex and locations were not statistically significant (P>0.05).
The prevalence of diesease was determined to be 10.5% (43/368) in sheep in 21
(44.7%) flocks, overall. It was 5.6% (4/71) in males and 11.5% (39/340) in female sheep. The
seroprevalence ranged from 6.7% to 20.0% in different locations. It was 10.4% (10/96) in sheep
between 0-2 years of age and 10.5% (33/315) in sheep older than 2 years. However, the
differences by age, sex and locations were not statistically significant either (p>0.05).
A prevalence of 7.8% (8/102) was obtained in the examination of human sera. The
highest prevalence was seen in farmer and abattoir workers with 12.0%. Seopositivity was not
detected in laboratory workers and veterinary students. Notably, seropositivity was detected in
animals of all positive farmers.
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129
Current features of Mediterranean Spotted Fever in Bulgaria
E. Aleksandrov, D. Mitov, B. Kamarinchev, N. Bogdanov
Military Medical Academy, Sofia, Bulgaria
138A
Cases of Mediterranean Spotted Fever /MSF/ were registered for the first time
in Bulgaria in 1948. Until 1970 the occurrence of the disease was sporadic with
benign clinical course - the total of 240 cases were registered. Followed a period for
20 years when there were no registered cases of the disease.
After 1992 cases of MSF have been increasing to get to 716 for 1996, 1008 for
1997, and 717 for 1998. The endemic characteristic of the disease from the past is
preserved.
Today`s MSF has quite a heavier course with possible exitus letalis, compared
to the disease in the sixties. At average 10.45% from the cases develop
complications. There is considerable difference in the intensity of the epidemic
process in different endemic regions. Occupational role of the disease occurrence is
diminished, compared to the past when cases of MSF arose from people living in the
villages and dealing with stock-breeding. Now cases arise mainly from people living in
towns. The ratio town dwellers/village dwellers is 4.4:1. The number of inhabited
places where new cases occur increases annually. High incidence and heavier clinical
course determines the new social-health significance of rickettsial infection.
Etiologic role of C. burnetii and SFG rickettsiae in atypical
pneumonia
E. Aleksandrov, M. Teoharova, B. Kamarinchev, D. Mitov, N. Bogdanov.
National center for infectious and parasitic diseases
The Balkans are endemic for the most widely spread all over the world
rickettsioses: Q-fever and SFG rickettsiae. It is known that for the region
Medeterannean spotted fever /MSF/is typical. For a period of 5 years /1993 - 1995/ a
study of the etiological role of C. burnetii in atypical pneumonia was carried out in
Bulgaria. It showed that 15-25% from them at average were Q-rickettsiosis. For the
different years its relative part ranged from 12.48% /1995/ to 28.83% /1993/. During
an epidemic it reached 40.50% /August 1993/. Q-rickettsial pneumoniae were
established during the whole year. All age groups were involved. The risk factor was
wiped out. During the study period 10 chronic Q-rickettsial endocarditis were
established. C. burnetii was isolated from the valves of 3 operated patients. The
presence of rickettsial infection in sick persons with endarteriitis obliterans and
thrombosis of arteria femoralis is of particular interest. Antibodies against C. burnetii /I
and II phase/ were established in 44.41%, and against SFG rickettsiae in 2.90%.
In the control group /sick persons with varices, angioneuroses and
other/antibodies against C. burnetii were established in 3.12%, against SFG
rickettsiae in 0.39%. Etiologic treatment resulted in clinical improvement and
decreasing of antibody titers.
The number of cases with MSF has increased after 1994. During the acute
phase of the disease different complications are observed and the main part of them
comprises of cardio-vascular disorders /infections, attacks, thrombophlebitis,
myocarditis, pericarditis, and other/.
- 66 -
138B
A 15 year survey of anti-Rickettsia serology and clinical results in
a Belgian university hospital
141
G. Bigaignon, A. Crosiers
UCL University Hospital St Luc, 1200 Brussels.
Since September 1983, our Serology Laboratory has used in routine the
BioMérieux IFAT methodology with specific slides for Rickettsia conori, Rickettsia
mooseri and Coxiella burnetii.
On 31 December 1998, 19,392 analyses had been carried: 6,476 for Rickettsia
conori, 6,287 for Rickettsia mooseri and 6,629 for Coxiella burnetii.
Six patients were found to be positive for IgM and IgG anti-Rickettsia conori, all
of them with fever and a typical "tache noire" in August or September: one boy after
holidays in Provence, one boy coming back from Spain, one boy and one student
returning from Morocco and one couple infected in Belgium, probably by a tick from
their dog, also serologically positive.
The Rickettsia mooseri serology was very useful in confirming the suspicion of
murine typhus in two aduits, one of them with the professionnal sponsability to remove
dead rodents in the Southern part of Belgium. A seroconversion for IgM and IgG
occured also in a 58 year old male patient, one week after a 15 day journey in
Vietnam, Kampuchea, and Thailand : the clinical aspect was perfectly consistent with
a Rickettsia tsutsugamushi infection, with strong positivity with OX K Weil Felix
reaction.
For Coxiella burnetii, IFAT revealed presence of IgM and IgG in 8 febrile
patients: 3 veterinarians, 3 workers in a slaughterhouse, 1 coming back from a trip in
the Canary Islands and 1 bitten by a tick in Belle-Isle-en-Mer, in Brittany.
Carrion’s disease: findings of Bartonella bacilliformis cases from
the jungle of Peru
H. Vizcarra F., A. Tejada, J. J. Miranda, O. Palacios, A.L. Cuadra, J. Perez
Instituto de Medicina Tropical. DAC, Universidad Nacional Mayor de San Marcos.
Iglesias 582, Miraflores. Lima Perú
Bartonella bacilliformis causes Carrion’s disease in Peru, Ecuador y Colombia.
The bacteria is located inside of red cells during the anemic period and after an
angioblastic, warty, is developed in the cutanous period. The reservoir the human
beings and bacteria is transmitted by a sandfly: Lutzomyia; usually andean vallies of
the country we are performed an epidemiological survey in Monzon valley located in
the jungle area of departament of Huanuco in the central region of the country where
some clinical cases were reported in the last years.
Eigth hundred sixty blood smears were taken from the general population, 1.8
% showed Bartonella in the samples, one hundred nineteen blood cultured-Columbia
medium modified, were perfoamed from the same number of sick persons with ferver
and anemic 30% were positive. Lutzomyia serrana was the most often sanfly
founded.Our result confirm the presence of the Carrion’s disease in the peruvian
jungle as new area of the disease.
- 67 -
142
Comparative study of sera tested for Rickettsia antibodies by
Immunofluorescence staining, PanBio EIA and INDX Dip-S-Ticks.
1,2
1,2
2
150
1,2
Zoltan Nack* , Ling Wang , John Stenos and Stephen Graves
1 PathCare Consulting Pathologist, Ryrie St, Geelong, Victoria, 3220, Australia.
2 The Australian Rickettsial Reference Laboratory, The Douglas Hocking Medical
Institute, The Geelong Hospital, Geelong, Victoria, 3220, Australia.
We are currently analysing over 200 serum specimens submitted for rickettsial
serology in 1998, using 3 different testing procedures. For routine testing and as a
“gold standard” we use immunofluorescence (IF) staining, using anti-human
fluorescence labelled total antibody (IgM and IgG) against all 3 group of Rickettsia
(Spotted Fever, Scrub Typhus and Typhus group). Rickettsial species tested include
R. australis, R. honei, O. tsutsugamushi (Gilliam and Litchfield strains), R. typhi and
R. prowazekii. All sera were tested with a PanBio Spotted Fever Group enzyme
immunoassay (EIA) (IgM) and the INDX Dip-S-Ticks (DST) (R3LD) test, which is a
solid-phase EIA technique for the detection of antibodies to all 3 rickettsial groups.
Preliminary data indicates that both the IF and EIA assays shared similar specificities
which were slightly greater than the DST assay. The IF assay did however display
the greatest sensitivity followed by the EIA and the DST assays.
Studies on natural foci of Tsutsutsugamushi disease of the
autumn-winter type in Jiangsu
Guo Hengbin,Tang Jiaqi,Wu Guanghua
Institute of Military Medicine,Nanjing Command,PLA,Nanjing 210002
Before 1986, tsutsugamushi disease was known only prevalent in south to
Zhejiang Province in our country, belonged to the summer type and Leptotrombidium
(L. ) deliense was regarded as the main vector. In Nanjing and carried out a series of
studies in 1986¡«1992.The resultswere as follows: Tsutsugamushi disease was
epidemic in Nanjing and north of Jiangsu (including; Dongtai, Haian, Rudong, Jinhu,
Hanjiang, Jiangdu); belonged to the autumn-winter type; the main reservoir hosts
were Apodemus agrarius, Rattus confucianus, R. norvegicus and Crocidura lasiura;
the transmitting vector was L scutellare; the pathogen of tsutsugamushi disease of the
autumn-winter type, Rickettsia tsutsugamushi belonged to low-virulent strain, and
could not easily be detected; after the inoculated mice were treated with diluted
cyclophosphamide solution , 14 strains of R. tsutsugamushi have been isolated from
rats, mites and patients; serological typing of their sera showed that they belonged to
the Gilliam type; natural foci in Jiangsu could be divided into two types-flat land and
hilly land.
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165B
Serological evidence of human infection with Bartonella henselae
in Greece.
167
St.Alexiou Daniel, A.Tea, A. Papoutsi, A. Antoniadis.
Dept. of Microbiology, School of Medicine, Aristotelian University of Thessaloniki.
During the last year blood samples single or paired obtained from 178 patients,
children and adults, were examined for the detection of IgG specific antibodies to
Bartonella henselae by immunofluorescence. The symptoms of these patients were:
regional lymphadenopathy and fever (84 patients), fever of unknown origin (48
patients), fever with rash (13 patients), respiratory tract disease (28 patients), and
pericarditis (5 patients). In 13 out of 84 patients with lymphadenopathy a single high
titre 1 :128 of antibodies was found, whereas in two patients a seroconversion was
observed. In five of these cases a contact with domestic cats was reported.
Further on a single high titre of antibodies  1:128 was detected in 7 out of 48 patients
with fever of unknown origin, in 1 out of 13 patients with fever and rash, in 4 out of 28
patients with respiratory tract disease and in 1 patient with pericarditis. A
seroconversion was also found in a patient with pneumonia.
These preliminary data suggest that human infection with Bartonella henselae
in Greece is rather common.
Detection of Spotted Fever Group Rickettsia by half nested
polymerase chain reaction
YuQiang ChenXiangrui Zhangyongguo et
Institute of Microbiology and Epidemiology Beijing, 100071
We design three primer from 120kDa antigen gene of Rickettsia rickettsii to
detect Spotted Fever Group rickettsia by half nestle Polymerase Chain reaction. The
target sequence of rickettsia DNA was detectable as the band corresponding to 361bp
in 10fg of DNA extracted from yolk sac of embryonated Hen eggs infected rickettsiae,
and it was detectable to one rickettsia particle, we screened four method of prepared
template and found that method of glass milk is the simple, convenient. The PCR
result of template prepared by glass milk method is stable. The primers amplified
fragments of the predicted size from all spotted fever group rickettsiae (R. rickettsii, R.
parkeri, R. conorii, R. sibirica, R. akari, R. Jinghe strain, R. Heilongjiang 054 strain).
No amplified products were detected when R.. typhus group(R. canada, R.
prowazekii, R. typhi), R. tsutsugamushi, and Q Fever were assayed. In a study
experimentally infected guinea pig, the PCR method could detect rickettsia DNA from
4 days after inoculation(DAI). Although clinical manifestions subside after 13 DAI,
rickettsial DNA in blood samples could be detected by PCR for up to 30 DAI.
- 69 -
172B
Single-tube nested polymerase chain reaction assay for detection
of granulocytic Ehrlichia
177
Mauel, MJ, TN Mather
Center for Vector-Borne Disease, University of Rhode Island, Kingston, RI, USA
Nested PCR systems are utilized to increase sensitivity of PCR-based assays.
However, after the first amplification the tubes must be opened in order to transfer
template molecules to the second amplification reaction. The transfer procedure
inherently carries the risk of contamination from positive samples to negative samples.
To over-come this risk of contamination, a nested PCR assay for detection of
Granulocytic Ehrlichia in which both amplifications are performed in a single tube was
developed. The assay is based on 16S rDNA gene sequence with previously
determined species-specific primers. The single-tube nested PCR system has proved
to be as sensitive as the two-tube nested PCR assay. The assay was further modified
to reduce the cycling times reducing the denaturation, annealing and elongation times
to 10 seconds each greatly reducing the time needed to complete the assay. The
reduced amount of materials and time needed for the single-tube nested PCR greatly
reduces the cost of the assay. This single-tube nested PCR system should be a useful
addition to the diagnostic assays for Granulocytic Ehrlichia.
Study on Seroepidemiological Investigation of Spotted Fever in
Qiongzhong Area Of Hainan Province
Chang Binggong 1, Zhang Jianzhi 1,Tian Xiaodong 2,Lu Zhengzhi2
1 Institute of Microbiology and Epidemiology, Chinese Academy of Preventive
Medicine
Beijing,102206 - 2 Military Medical Institute of Guangzhou, Guangzhou 510507
In order to umderstand an epidemic situation of spotted fever in Hainan
province, serosurvey and kinds of rodent and their tick-carriing were carried out by
using CF in Dafeng¡¢Yangjiang and Xinjin Counties in Hannan Province.The results
showed that prevalence of antibodies to R.sibirica , R.conorii and R.akari were
4%(2/50)¡¢ 2%(1/50) and 2%(1/50) respectively in sheep£» 3.17%(2/63)¡¢ 0%(0/63)
and 3.17%(2/63) respectively in pig£» 16.67%(12/72)¡¢ 11.11%(8/72) and
2.78%(2/72) respectively among healthy persons£»and 30% (36/120)¡¢10.83%
(13/120) and 7.50% (9/120) respectively in wild mice£»there was no antibody to
spotted fever group rickettsiae to be found in cattle serum. The major wild mice are
R.losea, R.rattus hainanieus and R.fulvescens, the tick-bearing rate were respectively
11.91% (7/44)¡¢ 30.00% (15/50) and 36.36%(8/22).
- 70 -
180
Epidemiological and clinical features of Bartonella endocarditis : a
case control study
199
P.E. Fournier, H. Lelievre, S.J. Eykin, J.L. Mainardi, T.J.Marrie, D. Raoult.
Unité des Rickettsies, Faculté de Médecine, 27 Bd J. Moulin, 13385 Marseille Cedex
5, France
Bartonella are emerging pathogens responsible for blood culture-negative endocarditis.
However, due to the small number of reported cases, the epidemiological and clinical
characteristics of Bartonella endocarditis have yet to be evaluated. From 1995 through 1998, 64
patients were diagnosed in our laboratory as having Bartonella endocarditis on the basis of
serology and/ or isolation or PCR-amplification from blood or valvular biopsies. 84.4 % of these
64 patients had antibody titers to Bartonella spp. > 1:1,600 and the pathogen was isolated or
identified by PCR and sequencing in 34 patients, B. henselae in eight (23.5 %) and B. quintana
in 26 (76.4 %). In order to characterize the species-specific epidemiological and clinical features
of Bartonella endocarditis, we compared those of the eight patients with B. henselae
endocarditis and the 26 patients with B. quintana endocarditis with 16 and 52 control patients,
respectively, matched by sex, age and geographic origin, randomly selected among patients
with infective endocarditis of other etiology in a nested case-control study. Epidemiological and
clinical data were obtained by means of a standardized questionnaire including the Duke
criteria for the diagnosis of infective endocarditis. Patients with B. henselae endocarditis were
as likely as their controls to have a previous valvular abnormality (p = 0.32) and were
epidemiologically linked to cat bites or scratches (p = 0.001) and cat flea exposure (p = 0.0007)
whereas those infected with B. quintana were less likely than controls to have a previously
known valvular disease (p = 0.001, OR = 0.2) and to be immunocompromised (p = 0.02, OR =
0.1), but were more likely to be homeless (p < 10-5, OR = 96.3), alcoholic (p = 0.00002, OR =
9.0) and exposed to body lice (p = 0.0001). Our findings demonstrate that distinct epidemiologic
risk factors are associated with B. quintana and B. henselae endocarditis. In patients with
culture negative endocarditis, B. quintana should be highly suspected in homeless and
alcoholic patients with no previous cardiac history whereas B. henselae should be suspected in
patients with a valvulopathy in contact with cats and cat-fleas.
Samples dried on blotting paper to diagnose rickettsial diseases.
F.fenollar1, D. raoult1
1Unité des Rickettsies, Faculté de Médecine, CNRS UPRES-A 6020, 13385 Marseille,
France.
The use of filter-paper is an inexpensive and convenient method for collecting,
storing and transporting blood samples for serologies. In addition, samples occupy
little space, and can be readily transported without refrigeration. Rickettsial diseases
evolve often according to an epidemic mode and are now considered as re-emerging
diseases, especially in developing countries, under conditions where field work could
be difficult. The suitability of collecting whole blood specimens on filter paper disks for
rickettsial anticorps assay was evaluted. Blood specimens from 64 individuals with
antibodies to Coxiella burnetii, Bartonella quintana or Rickettsia conorii were tested for
rickettsial antibodies by microimmunofluorescence. Although occasional titres were
one or two dilutions lower, no significant differences where observed by statistical
analysis. Among patients with a negatif serology, no false-positives were found.
These results demonstrate that recovery of antibodies from fingerstick blood dried on
filter paper after elution is comparable to usual serum. Storage of paper samples for
one month at room temperature or at 4°C did not significantly affect the anticorps
level. This report shows the utility of this sample collection method in developing
countries when refrigeration is not possible and where venipuncture is a problem.
- 71 -
202
In vitro activity of a new ketolide antibiotic, HMR 3647, against
Rickettsia rickettsii, Rickettsia conorii, Rickettsia africae,
Rickettsia typhi, Rickettsia prowazekii, Coxiella burnetii,
Bartonella henselae, Bartonella quintana, Bartonella bacilliformis,
and Ehrlichia chaffensis.
Jean-Marc Rolain, Max Maurin, Mireille Sobraquès, and Didier Raoult *
Unité des Rickettsies - CNRS UPRES A 6020 - Faculté de Médecine - Marseille France
Ketolides are a new class of macrolide antibiotics that have been shown to be
active against various intracellular organisms including Legionella pneumophila and
Chlamydia trachomatis. In this study we have tested the bacteriostatic and the
bactericidal activities of HMR 3647 against Rickettsia rickettsii, R. conorii, R. africae,
R. typhi, R. prowazekii, Bartonella henselae, B. quintana, B. bacilliformis, Coxiella
burnetii and Ehrlichia chaffensis. HMR 3647 was more active than erythromycin
against Rickettsia, Bartonella, and C. burnetii with MICs of 0.5, 0.003 to 0.015, and 1
µg/ml, respectively. No bactericidal activity of HMR 3647 against these pathogens
was found. HMR 3647 did not display any bacteriostatic or bactericidal effect against
E. chaffensis. These results demonstrate that HMR 3647 possess interesting in vitro
activity against Rickettsia spp, Bartonella spp., and Coxiella burnetii and is potentially
useful for the treatment of rickettsiosis, acute Q fever, and bacillary angiomatosis
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204
Tuesday June 15th
Slide session 4
Diagnosis and Treatment of Rickettsioses
Hidden mortality attributable to Rocky Mountain spotted fever:
immunohistochemical
detection
of
fatal,
serologically
unconfirmed disease
CD Paddock, PW Greer, TL Ferebee, J Singleton Jr, DB McKechnie, TA
Treadwell, JW Krebs, MJ Clarke, RC Holman, JG Olson, JE Childs, SR Zaki.
Centers for Disease Control and Prevention, Atlanta, GA, USA.
Rocky Mountain spotted fever (RMSF) is the most severe tick-borne infection in
the United States and is a nationally notifiable disease. Since 1981, the case-fatality
ratio (CFR) for RMSF has been determined from laboratory-confirmed cases reported
to the Centers for Disease Control and Prevention (CDC). Serologic assays for
detecting antibodies reactive with Rickettsia rickettsii are the most widely available
and most frequently used methods to confirm a diagnosis of RMSF, but are frequently
non-diagnostic in the first 10 days of the disease. We describe 9 patients with fatal,
serologically unconfirmed RMSF for whom diagnoses of RMSF were established by
immunohistochemical (IHC) staining of tissues obtained at autopsy. During 19961997, acute-phase serum samples and tissues from patients with fatal disease
compatible with RMSF were tested at CDC. A median of 7 days (range, 5 to 10 days)
elapsed from onset of disease to death. Using an indirect immunoflourescence assay,
no patient serum demonstrated IgG or IgM antibodies reactive with R. rickettsii at a
diagnostic titer (e.g.,  64); however, IHC confirmed RMSF in all patients. PCR
validated the IHC findings for 2 patients for whom appropriate samples were available
for testing. These findings suggest that dependence on serologic assays and limited
use of IHC for confirmation of fatal RMSF will underestimate mortality and possibly
CFRs for this disease.
- 73 -
16
Laboratory diagnosis of rickettsial diseases in Queensland,
Australia, 1994 – 1998
3
Carmel Taylor
Queensland Reference Laboratory for Rickettsial Diseases, Brisbane, Australia.
The Queensland Reference Laboratory for Rickettsial Diseases was
established in 1994, and provides a reference service for rickettsial diseases for
Queensland (population 3.3 million) and communities in Northern New South Wales
(NSW). The rickettsial diseases known to be endemic to this region are Queensland
tick typhus (QTT, caused by R australis), murine typhus and scrub typhus. All sera
referred for rickettsial serology are screened by the indirect fluorescent antibody test
for antibodies to R. australis, R. typhi and O. tsutsugamushi. During the period 1994 1998, 92 patients were confirmed as having a rickettsial infection by demonstration of
a four-fold or greater rise in antibody titre. A further 137 patients were shown to have
significant antibody titres (>=256), although a rise in titre was not demonstrated. The
confirmed rickettsial infections included 44 cases of QTT (a further 115 patients had
significant titres to R. australis), a single case of murine typhus in a returned traveller,
and 47 cases of scrub typhus (22 further patients had significant titres). Apart from
two outbreaks of scrub typhus among military personnel in 1996 and 1997, rickettsial
infections in Queensland occur sporadically and in low numbers. The geographical
distribution of cases is suggestive of the existence of several foci of rickettsial
diseases throughout the state and adjacent areas. Most cases of QTT occur in the
Northern Rivers area of NSW, the Sunshine Coast area and the Cairns area, while
scrub typhus appears to be largely confined to several discrete localities in the far
north of the state.
Coxiella burnetii in ticks, goats, sheep, and humans in Cyprus:
Detection, Isolation and Molecular Identification of Six Strains.
Loukaides Fidias2, Psaroulaki Anna1, Spyridaki Ioanna1, Hadjichristodoulou
Christos2, Tselentis Yannis1.
Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical
Medicine, Faculty of Medicine, University of Crete 1, Heraklion, Greece, and Veterinary
services of Cyprus2 , Athalassa, Nicosia, Cyprus.
Coxiella burnetii is distributed world-wide and has a broad spectrum of hosts,
including ticks, domestic animals, man, rodents, and birds. Over a period of one year
(1996-1997) in Cyprus, we collected 142 ticks (Rhipicephalus sanguineus and
Hyalomma spp.) from goats and sheep, and obtained 17 blood samples from these
animals as well as humans. Ticks were screened for the presence of Coxiella burnetii
by the hemolymph test, whereas the blood samples were tested by the
immunofluorescence (IFA) technique. Using a centrifugation shell vial technique, one
strain of Coxiella burnetii was isolated from tick hemolymph, two strains from triturated
ticks, and three strains from blood using human fibroblast monolayers. The
microorganism was detected in the cultures by the IFA technique, the Gimenez stain,
and the cytopathogenic effect on Vero and HEL cells. Restriction fragment length
polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified gene
fragments was used to identify the six Cyprus isolates, and compare them with the
reference strains Nine Mile and Q212. In order to achieve a direct detection of
Coxiella burnetii in ticks and blood, a “nested” PCR was performed, resulting in 11 of
142 examined ticks and 8 of 17 blood samples positive for Coxiella burnetii.
Our study demonstrates that Coxiella burnetii is endemic in Cyprus,and that
direct detection of the microorganism in ticks and buffy coat can be accomplished
using a nested’’ PCR technique. The Cyprus strains are genotypically identical to the
reference strains Nine Mile and Q212.
- 74 -
144A
Seroepidemiology of bartonellosis in Poland
Stanislawa Tylewska-Wierzbanowska, Edyta Podsiadly
National Institute of Hygiene, Warsaw, Poland
23
In 1998 patients suspected for Bartonella sp. infections due to particular clinical
symptoms were tested serologically with indirect immunofluorescence assay (MRL
Diagnostics, USA). Among 72 patients tested in our laboratory, 32 were found
seropositive. Antibodies specific to B. henselae and B. quintana were detected in 20
and 4 of them, respectively.
Geographic distribution of cases and clinical symptoms, age, gender of patients
were analyzed.
Human Ehrlichiosis surveillance in the United States
Jennifer H. Mc Quiston, C.D. Paddock, R.C. Holman, J.E. Childs
Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases,
Centers for Diseases Control (CDC), Atlanta, GA
Ehrlichiosis is an emerging tick-borne zoonoses of increasing public health
concern. Human monocytic ehrlichiosis (HME) is caused by Ehrlichia chaffeensis, and
Human granulocytic ehrlichiosis (HGE) is caused by an agent similar or identical to E.
equi and E. phagocytophila. Other species such as E. ewingii may also represent
emerging zoonotic pathogens. In the United States, ehrlichiosis surveillance has been
hampered by the limited availability of diagnostic tests and the lack of an official
reporting system. A review of surveillance efforts made by state health departments
showed that ehrlichiosis was notifiable in 19 states in 1998, and that diagnostic tests
were offered by 12 state laboratories. Through 1997, 742 cases of HME and 449
cases of HGE were reported nationwide. The annual number of reported ehrlichiosis
cases rose sharply between 1994 and 1997. Annual incidence was calculated for 24
states which established surveillance systems. From 1986 through 1997, 827
confirmed and probable cases of ehrlichiosis were diagnosed by serologic testing at
CDC, although it is not known how many of these cases were reported by states. This
review represents the first comprehensive summary of state surveillance practices
and reported ehrlichiosis incidence in the United States, and underscores the need for
standardized national surveillance efforts.
- 75 -
94
Influence of occupation, pre-existing illnesses, and chronic
medication use on the severity of illness of human granulocytic
ehrlichiosis (HGE).
97
Johan S. Bakken, Robert L. Tilden, SMDC Health System, Duluth MN, and
Jennifer Walls, J. Stephen Dumler
The Johns Hopkins Medical Institutions, Baltimore, MD, USA.
To assess possible influence of occupation on risk of acquiring HGE and preexisting
illnesses and chronic medication use on the severity of illness, we reviewed the case records of
140 patients with probable or confirmed HGE. We considered hospitalized patients to have a
more severe illness. Prior diagnoses were categorized into groups, including malignancies,
immune-modulating illnesses, diabetes mellitus, and prior Lyme borreliosis, and medications
were grouped in a similar manner. The risk for tick-bites and HGE was correlated with inside or
outside occupation. All patients resided in the upper Midwest USA. P < 0.05 was considered
significant.
Patients who reported tick-bites were hospitalized less frequently than those who did not
(38.6% vs. 65.4%, rr 0.65, CI 0.44-0.84, p=0.003). The mean age and temperature were higher
for hospitalized patients (62.4 vs. 52.5 years, p=0.039 and 39.3 vs. 38.9 0C, p=0.050). More
severe illness was associated with pre-existing immunemodulating illnesses and immunesuppressive therapy (p=0.001), but not with a history of prior malignant disease, diabetes
mellitus, or concurrent antibiotic therapy. There was a trend towards less severe illness among
patients who had a history of previous Lyme borreliosis. There was no difference in hospital
admission frequency among patients employed outdoors or indoors. Five butchers, who did not
recall any tick-bites became infected after having sustained multiple cutaneous injuries while
cutting large numbers of deer carcasses during late fall hunting season.
Patients who have immune-compromising illnesses, or who receive immunesuppressive
medications may be at increased risk for severe HGE. Individuals who are exposed to large
volumes of body fluids (esp. blood) from infected mammals, such as deer, may be placed at risk
for acquiring HGE.
Immunodiagnosis of human granulocytic ehrlichiosis using a
recombinant HGE-44-based ELISA
Jacob W. Ijdo1, Caiyun Wu', Louis A. Magnarelli2, Erol Fikrig'
'Section of Rheumatology, Department of Internal Medicine, Yale University School of
Medicine, New Haven, Connecticut and 2The Connecticut Agricultural Experiment
Station, New Haven, Connecticut
Current antibody testing for human granulocytic ehrlichiosis (HGE) is performed
by using indirect fluorescent antibody (IFA) assays, immunoblots, or enzyme-linked
immunosorbent assays (ELISA). A major drawback of these techniques is the
variability of test results associated with the use of different strains of antigens derived
from horses or cultured HL-60 cells. We used recombinant HGE-44, expressed and
purified as a maltose-binding protein (MBP) fusion peptide, as antigen in a polyvalent
ELISA. Fifty normal sera from healthy humans were used as a reference to establish
a cut off level. Thirty-one of 35 (89 %) HGE patient sera, previously confirmed by
positive whole cell immunoblot, reacted positive in the ELISA assay. Serum samples
from patients with Lyme disease, syphilis and human monocytic ehrlichiosis did not
react with HGE-44-MBP. Larger sample sets need to be tested to determine
sensitivity and specificity values. We conclude that recombinant HGE-44 can be used
as an antigen in an ELISA for laboratory diagnosis of HGE.
- 76 -
101
Human Granulocytic Ehrlichiosis In Scandinavia
Anneli Bjöersdorff1, 2, Björn-Erik Kristiansen3, Ingvar Eliasson1
1Department of Clinical Microbiology, Kalmar County Hospital, Kalmar, Sweden 2
Department of Medical Microbiology, University of Lund, Lund, Sweden 3 A/S Telelab,
Gulset, Skien, Norway
109
In the Scandinavian countries granulocytic ehrlichiosis (GE) is a well known
disease in animals (dogs, horses, sheep and cattle) but so far only three human cases
have been presented. Serological data from tick-exposed populations indicate a
seroprevalence of approximately 4% in humans in Denmark, 10% in Sweden and
Norway. During 1996 – 1999 blood samples from patients with suspected human
granulocytic ehrlichiosis (HGE) in Sweden and Norway were analysed by a nested
granulocytic Ehrlichia specific PCR and serum samples were analysed by IFA for the
presence of antibodies to Ehrlichia equi. Twelve cases of laboratory-verified HGE
were found. Ten of them were PCR positive, two cases were PCR negative but
seroconverted or had a fourfold change in titre. The majority of the PCR positive
cases were seronegative at admission and some remained seronegative. Sequenced
PCR products showed homology with corresponding fragments of the 16S rRNA
genes of the “HGE agent” and with Ehrlichia genes sequenced from Swedish dogs,
cats and horses with GE. The HGE cases reflected wide variations in the clinical
spectrum of the disease but common clinical signs were fever, malaise, and
headache. Our results point to some possible pitfalls in the diagnosis of HGE and the
necessity of further evaluation and development of the diagnostic methods.
Bartonella bacilliformis in the Sacred Valley of the Incas, Cusco,
Peru, 1998
1
1
1
4
1
1
P.Ventosilla , M. López , A. Antúnez , R. Birtles , H.Guerra , J. Merello , B.
1
1
1
3
2
Infante , E. Pérez , C. Maguiña , M. Montoya , D. Raoult .
(1) Inst. Med. Trop. A. von Humboldt, UPCH (2)Unité des Rickettsies, Faculté de
Médecine, Université Aix-Marseille II, Marseille, France. (3)Hospital Regional,Cusco.
(4) Dep. Pathology & Microbiology, University of Bristol, UK.
Bartonella bacilliformis is the etiologic agent of Carrión’s Disease, endemic to
the South American Andes North of the Parallel 12, Latitude South. Since 1993, 3
patients are recorded from Cusco (Parallels 14-16). In May 1998 a large outbreak of
the disease in the Department of Cusco, initiated months earlier, was identified for the
first time by blood smears. Here we confirm, bacteriologically, 6 clinically diagnosed,
blood-smear positive patients from Urubamba and Calca, Cusco: Blood was cultured
on potato blood agar, 21-30°C; 1.0-1.5 mm white, shiny, convex colonies appeared
after 7 to 14 days, of Gram negative, oxidase negative, characteristically motile
coccobacilli, reacting (Indirect ImmunoFluorescence) with species-specific anti-B.
bacilliformis antibody. PCRs for Intergenic Transcribed Spacer (ITS) region, with DNA
from cultures yielded a single-sized amplification product of 1,000 base pairs, a size
indistinguishable from that of a B. bacilliformis positive control, and much smaller than
that of other species of the genus. Negative controls yielded no products. These
procedures, used here first, are being refined and modified for diagnosis and
epidemiology of this important disease; analysis of the heterogeneity of B.bacilliformis
isolates from different regions of Perú is ongoing.
- 77 -
135
Improved sensitivity of PCR for HGE using epank1 gene of E.
phagocytophila group ehrlichiae.
1
1
1
1
Jennifer J. Walls , Patrizio Caturegli , Kristin M. Asanovich , J. Stephen Dumler
1Division of Medical Microbiology, Department of Pathology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland
The agent of human granulocytic ehrlichiosis (HGE), E. phagocytophila, and
E. equi probably comprise variants of a single Ehrlichia species now called the
Ehrlichia phagocytophila genogroup. These species share a unique 153 kDa protein
antigen with ankyrin repeat motifs, EPANK1, encoded by a gene with at least 2 copies
in the HGE agent genome. Because of these features and the high degree of
conservation in the 16S rRNA gene currently used, the epank1 gene would be useful
for HGE PCR diagnosis. Primers for epank1 were synthesized flanking a region that
spans part of the 5’ ankyrin repeat region and part of the unique 3’ end. A total of 32
blood samples from patients with suspected HGE were tested with the epank primers.
All patients were previously tested by 16S rRNA gene (16S) PCR and IFA. 12
patients were 16S PCR-positive and had a seroconversion detected by IFA (group A),
10 patients were 16S PCR-negative but had a seroconversion by IFA (group B), and
10 patients were 16S PCR-negative and seronegative (group C). 10 of the 12 group
A patients were epank PCR-positive, all 10 of the group B seropositive patients were
now epank PCR-positive, and all of the PCR-negative and seronegative patients
(group C) were epank PCR-negative. The epank primers are more sensitive than the
previously used 16S rRNA gene primers and therefore, may be more useful in
diagnostic testing for HGE.
- 78 -
174
Wednesday June 16th
Poster session 5
Rickettsioses in Animals, Hosts and Vectors
Recombinant Antigens of Cowdria ruminantium
A.F. Barbet, S.M. Kamper1, W.M. Whitmire2, G.R. Reddy3, D. Mwangi4, M.J.
Burridge, A.M. Lundgren, A.L. Moreland, T.C. McGuire 5, F. Rurangirwa5, and
S.M. Mahan6.
University of Florida, U.S.A.; 4ILRI, Kenya; 5Washington State University, U.S.A.;
6UF/USAID/SADC, Zimbabwe. Present address: 1University of Miami, U.S.A.; 2Rocky
Mountain Laboratories, NIH, U.S.A.; 3Kansas State University, U.S.A.
C. ruminantium is an economically important rickettsial pathogen of ruminants present in
sub-Saharan Africa and the Caribbean. As yet, few antigen encoding genes have been
identified and analyzed for potential use in improved vaccines or diagnostic assays. In these
studies we employed a series of methods to rapidly identify additional genes of potential
interest. Two genomic libraries of C. ruminantium DNA were prepared from Gardel (Caribbean)
and Highway (Zimbabwe) strains by partial fill-in reactions in plasmid vector pGEM7Zf(+).
Individual, non-amplified recombinants were screened for reactivity with 1:4000 dilution of
serum from an immune sheep. 34 positive clones were identified. Most clones were also
recognized by T lymphocytes from cattle immunized by infection and treatment. Hybridization
screening of the 34 clones identified 4 each containing the known antigen genes map2 and
groE, one group of 3 cross-hybridizing clones, one group of 2 cross-hybridizing clones, and 21
unique clones. Insert DNA from representatives of each group and from most of the unique
clones was sequenced (a total of ~65kb). Proteins expressed from all cloned DNAs were also
analyzed by cell-free transcription and translation, and immunoprecipitation. These data
identified: a) numerous genes with significant similarity to those of Rickettsia prowazekii; b)
genes significantly similar to genes encoding outer membrane proteins of other prokaryotes; c)
unknown genes predicted to encode prokaryotic membrane lipoproteins; and d) DNA containing
unusual repeat structures (e.g. a gene containing 20 tandem repeats encoding a 9-mer
peptide). These analyses have identified new candidate recombinant diagnostic and vaccine
antigens for heartwater disease.
- 79 -
6A
Kinetics of Antibody Responses to the MAP 1B Antigen in Cowdria
ruminantium infected Bovines
1
1
2
3
12C
4
S. Semu , T.F. Peter , F. Jongejan , A.F. Barbet , D. Mukwedeya and S.M.
Mahan1
1,3 UF/USAID/SADC Heartwater Research Project, Zimbabwe; 2 University of Utrecht,
Netherlands; 3 University of Florida, Florida; 4University of Zimbabwe, Zimbabwe.
The kinetics of antibody production in cattle after Cowdria ruminantium infection
was analyzed with the recombinant MAP 1B indirect ELISA, which has been reported
previously to be the most specific serological assay for heartwater. Weekly sera over
12 months were analysed from 30 calves which were experimentally infected and
repeatedly exposed to C. ruminantium via laboratory-infected Amblyomma hebraeum
ticks. In addition, weekly sera over 14 weeks from 10 calves which were exposed to
field A. hebraeum tick-derived infection in a heartwater-endemic area of Zimbabwe
were analysed. MAP 1B specific IgM, IgG1 and total IgG antibodies developed with
similar kinetics in both laboratory and field infected cattle, peaking at 4-6 weeks post
infection and declining to baseline by 8-24 weeks. No further significant rises in IgG
and IgM levels were detected, despite repeated exposure to C. ruminantium via
infected ticks. IgG2 responses were not detected. Selected sera were re-analyzed
with a C. ruminantium antigen immunoblotting assay and an indirect
immunofluorescent test. Titres by both tests peaked between 5-9 weeks post
infection and declined to baseline, without further rises, by 30-45 weeks. These data
suggest that MAP 1B-specific antibody responses, and possibly others, are downregulated in cattle after infection, irrespective of repeated exposure to C. ruminantium.
Serological assays based on the MAP 1B antigen may, therefore, not be a reliable
indicators of C. ruminantium exposure in cattle, necessitating the use of other
antigens or direct detection tests such as PCR.
Validation and application of the Cowdria ruminantium-specific
pCS20 Tick PCR Assay
T.F. Peter1, B.H. Simbi1, A.F. Barbet2, A.R. Alleman2, M.J. Burridge2 and S.M.
Mahan1,2
1 UF/USAID/SADC Heartwater Research Project, Zimbabwe; 2 University of Florida,
Gainesville, Florida. USA.
The development and use of the pCS20 tick PCR assay for Cowdria
ruminantium, the causative agent of heartwater disease of ruminants, has been
reported previously (Peter et al., 1995). Here, the assay was further evaluated in
laboratory and field studies on the Amblyomma tick vectors. The assay detected DNA
of 37 geographically diverse strains of C. ruminantium and had a specificity of 98%.
The assay did not detect DNA of the closely-related agent Erhlichia chaffeensis.
Assay sensitivity varied with tick infection intensity and was high (97%) with ticks
bearing 107 organisms, but dropped to 28% with ticks bearing 102 organisms. When
applied to experimentally infected A. hebraeum ticks, PCR detected more infections
(93%) than the previous gold standard, the xenodiagnostic mouse inoculation assay
(8%) (kappa = 0.015, p=0.17). The PCR assay was also applied to A. hebraeum and
A. variegatum field -collected ticks from 17 heartwater-endemic sites in four southern
African countries. Cowdria ruminantium infection was detected by PCR in all of the
tick batches, while attempts at tick transmission of infection to small ruminants failed
with four of the batches. The pCS20 PCR assay is presently the best characterized
and most reliable test for C. ruminantium in ticks and will be a valuable tool in
epidemiological investigations of heartwater.
- 80 -
12D
Ehrlichia phagocytophila infection in lambs as a post mortem
diagnosis.
13
2
S Stuen', E Olsson Engvall .
Norwegian School of Veterinary Science, Department of Sheep and Goat Research,
Sandnes, Norway, 2National Veterinary Institute, Uppsala, Sweden
Sixty-four lambs that had died after a history of grazing on tick infested pastures
were investigated for an Ehrlichia phagocytophila infection, both by autopsy,
microscopic examination of stained spleen smears and a polymerase chain reaction
(PCR) technique. Of these lambs, 54 were found positive of a granulocytic Ehrlichia
infection by PCR (84%) and 44 were judged to be positive by pathological evaluation
(69%). The main pathological criterium of an E. phagocytophila infection was
splenomegaly. Only one out of the 44 lambs with an enlarged spleen was found
negative by PCR. However, 20% of the lambs that were positive by PCR had a
normal spleen. The present investigation indicates that samples from coagulated
blood, lung and spleen should be preferred for PCR analysis of suspected
granulocytic Ehrlichia infection in lambs. Bacteriological and histopathological
investigations indicated that at least 93 per cent of the granulocytic Ehrlichia infected
lambs had a secondary infection, the most common infection recorded was
septicaemia due to Pasteurella hemolytica (trehalosi).
Woodrats (Neotoma spp.) as reservoirs of granulocytic ehrlichiae
in the western United States.
William L. Nicholson 1, John W. Sumner 1, Martin B. Castro 3, Vicki L. Kramer 3,
Gary O. Maupin 2, and James E. Childs 1
1Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention,
Atlanta, GA; 2Bacterial Zoonoses Branch, Division of Vector-Borne Infectious
Diseases, Ft. Collins, CO; and the 3Vector-Borne Diseases Section, California
Department of Health Services, Berkeley and Sacramento, CA.
Specimens from dusky-footed woodrats (Neotoma fuscipes) and Peromyscus spp. (P.
maniculatus and P. truei) mice collected in northern California were tested by IFA for antibodies
reactive with the USG3 strain of granulocytic ehrlichiae (GE). Thirty-four percent of woodrats
were seropositive with titers ranging from 16-8192 (GMT=469), yet only 8% of Peromyscus
mice were IFA-positive. Polymerase chain reaction (PCR) assays of the groESL heat shock
operon were conducted on all seropositive and a subset of seronegative blood specimens.
While only one sample from Peromyscus mice was positive, ehrlichial DNA was amplified from
68% of the woodrat blood samples, but not from 40 seronegative samples. Subsequent
samples from re-trapped animals showed that blood remained positive by PCR for 1-8 months.
Both seropositive and PCR-positive woodrats were collected at each trapping session. Adult
and nymphal stage Dermacentor occidentalis and Ixodes pacificus were tested by PCR assay.
One of 84 Ixodes pacificus adults flagged from the site was found to harbor GE. Nucleotide
sequences from three woodrats were determined to be identical to each other over a 1256-bp
region, yet differed by one base from an E. equi sequence obtained from a California horse.
The sequence obtained from the tick differed by two bases from the woodrat sequences and by
one base from the horse sequence. In recent work, we have found serologic and molecular
evidence of GE in Mexican woodrats (Neotoma mexicana) collected near Ft. Collins, CO.
Studies are underway to evaluate the woodrat-associated tick, Ixodes spinipalpis (=I.
neotomae), as a vector among woodrats.
- 81 -
15
Domestic foci of the brown dog tick Rhipicephalus sanguineus
(Acari: Ixodidae) in Israel
24
Inria Ioffe-Uspensky*, Kosta Y. Mumcuoglu* & Igor Uspensky**
*Department of Parasitology, The Hebrew University-Hadassali Medical School,
Jerusalem, and ** A. Silberman Institute of Life Sciences, The Hebrew University of
Jerusalem, Jerusalem, Israel
Several cases of the establishment of domestic foci of Rhipicephalus
sanguineus, the main vector and reservoir of Rickettsia conorii, the causative agent of
Mediterranean spotted fever, were observed in Jerusalem. A single female tick fully
engorged on a dog that drops off in or near a human dwelling may initiate the
beginning of a focus. Two types of foci were noticed: in small yards or gardens near
the dwellings where dogs live in kennels (pseudo-domestic foci) or just inside
apartments, often in many-story buildings, where dogs are kept indoors. In these foci,
the development of the tick changes from the normal 3-host cycle to a 2-host cycle
when larvae do not leave the dogs after feeding but molt into nymphs which feed
again on the same host. This pattern of behavior obviously increases the speed of tick
development. Engorged nymphs leave the host and migrate upwards over the inside
or outside walls of the house, often forming clusters in small cracks or depressions
inside the walls. Ticks were also found all over the kennels and in small holes in the
soil next to the kennels. Certainly, such foci create an additional risk to humans who
can be attacked by infected ticks in and around their homes. Such foci might be the
cause of some cases of illness recorded from large towns of Israel.
Use of Pulsed-field Gel Electrophoresis to Differentiate between
Cowdria ruminantium Stocks from Different Geographical Areas
E. P. de Villiers, K. A. Brayton, E. Zweygarth, and B. A. Allsopp.
Onderstepoort Veterinary Institute, Onderstepoort, 0110, South Africa.
Cowdria ruminantium stocks from different geographical areas were compared
by restriction endonuclease digestion of chromosomal DNA with SmaI followed by
pulsed-field gel electrophoresis. Fewer than 10 fragments in the range 97-700 kb
were obtained, giving characteristic profiles or 'fingerprints' for the different stocks.
Summation of the estimated sizes of the fragments gave chromosomal size estimates
ranging between 2263 and 2394 kb for the different stocks, with an average of 2329
kb. The estimates for each stock were Welgevonden, 2285 kb; Pokoase, 2335 kb;
Sankat, 2394 kb; Mara 87/7, 2390 kb and Senegal, 2263 kb. All the stocks contained
an extra-chromosomal element migrating with an apparent size of 815 kb. The
Welgevonden, Mara 87/7 and Senegal stocks each showed a unique macrorestriction
fragment pattern, while the Pokoase and Sankat stocks had identical profiles that
were different from those of the other stocks. This suggests that the two latter stocks,
which were isolated from two locations approximately 20 kilometers apart in Ghana,
West Africa, may represent a single genotype. All the stocks could also be assigned
to clusters representing different geographical areas. The discriminatory power of this
technique is high since the profile is representative of the complete genome, it
therefore provides valuable epidemiological data and could lead to the development of
grnotype-specific DNA probes.
- 82 -
44
An investigation of Coxiella burnetii infection at calving within a
herd of dairy cows in England using PCR
1
2
2
3
4
102
5
G.A. Paiba , G. Lloyd , K. Bewley , G.J. Webster , L.E. Green & K.L. Morgan
1 Formerly Department of Clinical Veterinary Science, University of Bristol, Langford
House, Langford, Bristol BS40 5DU, United Kingdom. 2 Centre for Applied
Microbiological Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK. 3
Veterinary Investigation Centre, Veterinary Laboratories Agency, Langford, Bristol
BS40 5DU, UK 4 Department of Clinical Veterinary Science, University of Bristol,
Langford House, Langford, Bristol BS40 5DU, UK. 5 Department of Veterinary Clinical
Sciences, University of Liverpool, ‘Leahurst’, Neston, South Wirral L64 7DE, UK.
Coxiella burnetii is reported to be a highly infectious pathogen and infection in
people has been associated with cattle contact.
A Polymerase Chain Reaction (PCR), used for the detection of C.burnetii in
human endocarditis lesions, was modified and used to examine bovine placentae,
vaginal swabs and colostrum. Samples were collected from all the cows that calved
over a period of five months on one farm where an abortion was identified to have
been associated with C.burnetii.
Evidence of C.burnetii infection was demonstrated in 85% of the cows
examined. Shedding was most frequently detected using samples of placenta or
vaginal swabs taken at calving although 38% of colostrum samples and 13% of
vaginal swabs taken from non-pregnant cows were also PCR positive. The PCR was
more sensitive that microscopy.
The findings presented indicate that where C.burnetii is present in a dairy herd,
infection is disseminated widely among the apparently healthy animals. In
consequence, calving of these cows presents a period of high risk for those in close
contact. Advice is given to disposed of all placentae by incineration.
Field evidence that roe deer (Capreolus capreolus) is a natural
host for Ehrlichia phagocytophila
Alberdi, M. P., Walker, A. R., and Urquhart, K.
Samples of blood, spleen and ticks from 112 culled roe deer (Capreolus
capreolus) were collected from nine widespread sites in the United Kingdom. The
prevalence of infection with E. phagocytophila determined by means of serology and
polymerase chain reaction. Means of 58% of 102 plasma/serum samples were
seroreactive by IFA, 38% of 84 blood and 29% of 82 spleen samples were positive by
PCR. Ticks on 70 deer legs were all Ixodes ricinus larvae, nymphs and adults and
59/70 animals (84%) were infested. Numbers of ticks corresponded positively to the
percentage of samples positive for E.phagocytophila by serology and PCR for each
area. Ixodes ricinus nymphs collected from the vegetation at one site with infected
deer were examined for infection with E.phagocytophila by examination of Feulgen
stained salivary glands. Of 135 nymphs 5% were infected. These results confirm that
the roe deer is commonly parasitized by both E.phagocytophila and its vector tick in
such a way that it is likely to be the natural mammalian reservoir of E.phagocytophila.
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122
Serologic monotoring of zoonosae
Ribakova N*, Tokarevitsch N**
*: Regional Centre of State Sanitary and Epidemiologic Supervision - **: St Petersburg
Pasteur Institute, Russia
123
Anthropogenic influence upon natural foci of diseases, reforming of national
economy, real danger of uncontrolled delivery of agricultural animals froms States of
Cummunity and regions of Russia unfavourable for diseases common for people and
animals, demands intensification of epidemiologic supervision on zoonosae. One can
get objective information about pathogen circulation in nature and population, about
intensity of epidemiologic process and real infection spreading on concrete territory
among different age groups and heightened risk infestation contingens by studying
immunologic structure of population. It is especially important for developing and
improving epidemiologic supervision on diseases, those are not under epizootological
control of veterinary service : leptospirosis and Q fever.
Personnel of cattle-breeding farms and enterprises working over output and raw
having animal origin were taken as an object for supervision. The basis of selection
criterion for indicator group making up serologic monitoring is the difference between
immunologic indices (p<0.05) of persons professionally connected with cattlebreeding (leptospirosis - 10.9% , Q fever - 4.5%), and control groups : urban and
village population that is not professionally connected with agricultural animals
(accordingly 6.3% and 3.1%).
Sequence Analysis of the romp B Gene of Rickettsia prowazekii
Isolated from Flying Squirrels
Cecilia G. Moron, M.D., Xue-Jie Yu, M.D., Ph.D. Patricia Crocquet-Valdes, M.S.
and David H. Walker, M.D.
Department of Pathology, and WHO Collaborating Center for Tropical Diseases,
University of Texas Medical Branch, Galveston, Texas.
Rickettsia prowazekii has been isolated from flying squirrels, Glaucomys
volans, in Florida and Virginia. We undertook sequencing and molecular analysis of
the rompB gene from R. prowazekii isolated from these flying squirrels and a
comparison with its homolog in R. prowazekii Madrid E and Breinl strains. The genes
of the Florida and Virginia strains have regions, partially sequenced, with more than
99% similarity to R. prowazekii Madrid E. However, when compared to R. prowazekii
Breinl, we detected a difference of 12 nucleotides out of 1059 in the Florida strain, and
13 nucleotides out of 1060 in the Virginia strain at the 5’ portion of the gene. We
suspected a sequence error for the R. prowazekii Breinl rompB gene, so we amplified
and sequenced the gene. Our preliminary sequencing data confirmed that there were
nucleotide errors in the remitted sequence. These results indicate that there seems to
be a close similarity in the sequences of R. prowazekii isolated from flying squirrels
when compared to Madrid E and Breinl strains for the portions analyzed thus far.
Sequencing of the entire romp B gene for the R. prowazekii Breinl strain and the flying
squirrel strains from Florida and Virginia is in progress.
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158
Ehrlichia canis DNA found in ticks in south of China
Pan Hua(1) Cheng Xiangrui(2) Ma Yuhai(1) Sun Yang(1) Yu Qiang(2) Zhang
Xueying(2) Niu Hua(2) Zhang Yongguo(2) Jacqueline Dawson(3)
The Military Medical Institute in Guangzhou Area, Guangzhou 510507(1), Institute of
Microbiology and Epidemiology of Beijing 100071 (2) ,Centers for Disease Control
and Prevention National Center for Infectious Disease(3)
164A
Ehrlichia is one of the endangerment zoonoses, almost global distribution,
particularly in tropical and subtropical regions. There is increased public awareness of
the disease since new species have been found in recent years. It is evidence that, by
present, ticks are the major vectors of the agent of Ehrlichiosis.Our country has a vast
territory and a great variety of ticks, and human being and animals are easily exposed
to ticks. There are some indications that there may by Ehrlichiosis in our country
though it has not been reported by now. We detected the tick samples collected from
Guang Dong, Guang Xi and Hai Nan province by polymerase chain reaction using
primers for Ehrlichia-genus specific portion of the 16S rRNA gene. The characteristic
452-bp product of Ehrlichia-genus was amplified from Rhipicephalus sanguineus
obtained from Guang Dong and Boophilus microplus obtained from Guang Xi .
Specific primers for Ehrlichia canis were used to amplify these samples again, and
characteristic 555-bp product was observer. The DNA fragments amplified were
cloned and sequenced. The result was submitted to the National Library of
Medicine/National Institutes of Heath of America and compared with 354,937
sequences from GenBank, EMBL, DDBJ, PDB nucleotide database. The most
similarity showed between the DNA fragment obtained from two species of tick and
Ehrlichia canis. The nucleotide sequence of the fragments differs in one to four
position compared with Florida and Oklahoma strain. This is the first time to find the
pathogenic evidence of the existence of Ehrlichia cains in our country. It indicates that
there may be nature infective spots in our nation. More researches are needed.
Canine ehrlichiosis found in China
Pan Hua(1) Ma Yuhai(1) Tong Shide(1) Sun Yang(1) Wen Bohai(2) Cheng
Xiangrui(3) Yuqiang(3).
The Military Medical Institute in Guangzhou Military Area, Guangzhou 510507(1)
Department of microbiology, The third Military Medical University, Chongqing
400038(2) Institute of Microbiology and Epidemiology of Beijing 100071(3)
An outbreak of canine epidemic disease has occurred at a canine feed lot in a
Guangzhou suburb since April, 1998. The infected dogs were adult German shepherd
dogs and Rottweiler. The infestations were most common during July through August.
The main clinical signs of the disease included fever, epistaxis, anemia and
leukopenia. Penicillin and gentamycin failed to cure the disease. Counting up the
number of infected animals by epistaxis, the cases were twenty, with six fatalities.
We got involved in diagnose at the beginning of September. Whole blood samples
(2ml) of three infected dogs were inoculated intravenously into three healthy 2months-old Beagle. The dogs run fever by 10 to 20 days post-inoculation, and
leukopenia, anemia, emaciation followed. The course of disease had gone on for one
to two months, and all of them died. Liver and spleen were swelling, by 2 times, and
rough surface by postmortem examination. White blood cell samples from the
primary and inoculated infected dogs were detected by polymerase chain reaction.
Primers , which amplify the 16S rRNA gene of ehrlichia species other than
E.sennetsu ,were used. Specific fragments of 452 base pairs (bp) were amplified
from all of the samples. It had been confirmed by the result of DNA sequencing in a
variable region of 16S rRNA gene that the agent of the disease was Ehrlichia canis.
This is the first time to fine the existence of canine ehrlichiosis in China. The work of
isolating the agent is being done.
- 85 -
164B
Immunopathologic evaluation of B. Vinsonii infection in dogs
Brandee Pappalardo, Talmage Brown, Mary Tompkins,
Breitschwerdt
North Carolina State University, College of Veterinary Medicine
and
Edward
175
An extensive experimental infection study was conducted in 12 SPF beagles. Six control dogs were
injected IV with sterile saline and six dogs were inoculated IV with 109 cfu of B. vinsonii subsp. berkhoffii.
All six dogs infected with B. vinsonii seroconverted and became bacteremic. When compared with control
dogs, flow cytometric analysis of peripheral blood cells revealed a generalized depression of CD8+
lymphocytes in dogs chronically infected with B. vinsonii. Furthermore, the cell surface phenotype of CD8+
cells differed between control and infected dogs. Fewer CD8+ lymphocytes from infected dogs were found
to express CD62L (L-selectin), CD49D (VLA-4), and CD11A (LFA-1). These findings may indicate impaired
homing to and activation within lymph nodes and impaired migration to sites of inflammation. Therefore,
CD8+ lymphocytes in Bartonella infected dogs may represent an immunologically impaired subset of cells.
Flow cytometric analysis of lymph node cells from the experimentally infected dogs was also evaluated.
When compared with control dogs, lymph node cells from Bartonella infected dogs showed increased levels
of T cells. The increased number of T cells was reflected in the CD4+ subset, which contained an elevated
number of cells expressing CD45RA and CD62L (markers indicative of a naive phenotype). Additionally,
fewer B cells were found to express cell surface MHC II in B. vinsonii infected dogs. It appears, then, that
an active cell-mediated immune response is mounted in dogs infected with Bartonella. This response likely
results in increased migration of naive CD4+ T cells into various lymphoid tissues including peripheral
lymph nodes, however, antigen presentation to these T cells may be impaired.
In light of these findings, correlation of immune status with severity of disease seems particularly
relevant. If, similar to our observation in dogs, CD8+ lymphopenia accompanies human infection with
Bartonella, particularly in CD4+ T cell depleted AIDS patients, the added immunosuppressive effect might
contribute to enhanced predilection for other bacterial or viral infections or cancer. The role of Bartonella in
induction of immunosuppression is not unfounded. Varying degrees of immunosuppression have been
described following infection with B. bacilliformis resulting in infection with opportunistic pathogens of
bacterial, parasitic and viral etiology which have been shown to complicate this disease.
Anti-tick antibody detected in dogs for an epidemiological study of
Ehrlichia canis infection in Yamaguchi, Japan.
Inokuma, H., Ohno, K. and Onishi, T.
Laboratory of Veterinary Internal Medicine, Yamaguchi University, Yamaguchi 7538515, Japan
Rhipicephalus sanguineus, a well known vector of Ehrlichia canis, is not
common in Japan except for in tropical Okinawa. in Yamaguchi Prefecture, the ticks
have never been found, however, 5 % of dogs were found to be positive for the
antibody against E. canis. The reason why positive dogs for E. canis exist without R.
sanguineus is unknown. Antibodies against 24kd protein from R. sanguineus (Rs24p)
were specifically produced after repeated infestation with adults (Inokuma et al. 1999).
in this study, some features of an anti-Rs24p antibody detected by ELI SA and the
relationship between the anti-tick antibody and E. canis infection were examined.
Canine sera were collected from 4 groups, (Ex) 3 dogs which underwent
experimental infestation with adult ticks repeatedly, (P) 57 positive control dogs
naturally infested with the ticks in Okinawa, (N) 30 negative control dogs and (Ec) 22
E. canis antibody positive dogs in Yamaguchi. The peroxidase-ABTS substrate
system was used for the ELI SA then the optical density (OD) of each dog was
measured at 405 nm.
The OD of dogs in group Ex was increased after the second infestation. There
was a significant difference of mean OD values between group P and N. These
results suggested that anti-Rs24p anti body detected by ELI SA is an epidemiological
marker of repeated tick exposure. There was no significant difference in the mean OD
value between group N and Ec. However, some dogs infected with E. canis showed
higher OD values compared~with the mean OD of tick negative dog s. R. sanguineus
may be reiated to the E. canis infection in Yamaguchi. We thank Dr.S.Yamamoto,
Azabu University, for identifying the E. canis antibody.
- 86 -
184
Preliminary observations on the seroprevalence of Q fever
(Coxiella burnetti ) in domestic animals in Mali.
203
Baradji.I, Brouqui, P.
Laboratoire Central vétérinaire, Bamako Mali & Unité des Rickettsies, CNRS
UPRESA 6020 Marseille, France
Studies on Coxiella burnetti conducted on human blood donors in Bamako
(Mali) indicated a seroprevalence of 24%. Cattle and sheep are the commonly
recognized reservoirs for C burnetii infections in humans. To establish the
responsability of both cattle and sheep as potential reservoir of C burnetii a
serosurvey was then undertaken at the Central Veterinary Laboratory in Mali. A total
of 244 sera obtained from cattle and 129 from sheep were then tested. Cattle sera
were obtained from herds kept under a variety of environmental conditions and
husbandry systems whereas sheep sera were collected from small herds kept in
houses in Bamako. An Indirect Fluorescent Antibody (IFA) test was used to evaluate
the presence of antibodies reactive to Coxiella burnetti in sera. All cattle sera tested
were negative whereas 32% of sera obtained from sheep had C burnetii antibodies.
These results indicate that sheep but not cattle is likely to be the reservoir for Coxiella
burnetti in Mali.
Survey of horses from south-east France for antibodies reactive
with the agent of human granulocytic ehrlichiosis.
B. Davoust1 , P. Brouqui2, F.Pons1, M. Boni1, J. Bardiès3, D. Raoult2.
1Groupe de Secteurs Vétérinaires de Marseille, 2Unité des Rickettsies, Faculté de
Médecine de Marseille, 3Clinique Vétérinaire Equine de la Côte d'Azur, Cagnes-surMer, France.
Horse serums collected from 5 South-East French departments were tested for
seroreactivity with a cultured isolate (USG3) of the agent of human granulocytic
ehrlichiosis (HGE).
Of the 114 samples tested, 30 (26 %) were found to be reactive at IFA titers of
 25 (anti-IgG).
Seroreactive horses were found in the Alpes-Maritimes (42% of 12), Vaucluse
(28 % of 53), Bouches-du-Rhône (26 % of 19), Var (25 % of 8), and South Corsica (14
% of 22).
The antigenic relationship between the agent of HGE, Ehrlichia equi and E.
phagocytophila indicates that for the first time in France, horses have been in touch
with E. equi.
Until now equine ehrlichiosis (E. equi) had been described in the USA, Canada,
the United Kingdom, Switzerland, Germany, Denmark and Sweeden. Only the E.
phagocytophila bovine infection (tickborne fever) had been described in West France.
When the blood samples were taken, there were neither relations with Ixodes
sp. parasitism nor clinical signs.
The next studies will try to underline E. equi in the blood of suspicious horses,
in ticks and in any other reservoirs.
- 87 -
232A
Survey of seroprevalence of Bartonella vinsonii, Ehrlichia canis
and Coxiella burnetii infections in dogs in southeast France,
French Guyana, Martinique, Senegal, Ivory Coast and Sudan.
232B
B. Davoust1, M. Drancourt2, M. Boni1, D. Parzy3, J. Seignot1, V. Roux2, D.
Raoult2.
1Groupe de Secteurs Vétérinaires de Marseille, 2Unité des Rickettsies, Faculté de
Médecine de Marseille, 3Institut de Médecine Tropicale du Service de Santé des
Armées, Marseille, France.
Canine serums (n = 483) collected in 1997 and 1998 from Southeast France (n = 352), French
Guyana (n = 19), Martinique (n = 7), Senegal (n = 42), Ivory Coast (n = 12) and Sudan (n = 51) were tested
for seoreactivity (immunofluorescence tests) of Bartonella vinsonii, Ehrlichia canis and Coxiella Burnetii
infections. The dogs were kenneled at military establishments apart from Sudan native dogs.
Serologies of Bartonella vinsonii were positive ( 1/25) of 66 dogs (14 %) :4.8 % in the Southeast of
France, 21 % in French Guyana, 14 % in Martinique, 26 % in Senegal, 0 % in Ivory Coast and 65 % in
Sudan.
Serologies of Ehrlichia canis were positive ( 1/20) of 82 dogs (17 %) : 7 % in the Southeast of
France (only in Corsica), 0 % in French Guyana, 28 % in Martinique, 54.7 % in Senegal, 25 % in Ivory
Coast and 96 % in Sudan.
Serologies of Coxiella burnetii were positive ( 1/50) of 46 dogs (9,7 %) : 9.8 % in the Southeast of
France, 5.2 % in French Guyana, 0 % in Martinique, 11.6 % in Senegal, 8.3 % in Ivory Coast and 7.8 % in
Sudan.
No cross-reaction were noted. The B. vinsonii and E. canis infections are more frequently found in
the dogs parasited by Rhipicephalus sanguineus (Chi2-test p < 10-8). On the other hand, the PCR (E. canis
and B. vinsonii) carried out on the ticks from dogs in Sudan was negative. The C. burnetii infection is more
frequent in the dogs having contact with ruminants (Chi2-test p < 10-6).
This results confirm the hight prevalence of canine ehrlichiosis in Africa. The dog can be a reservoir
of B. vinsonii and C. burnetii. The PCR analysis must clarify the role played by R. sanguineus in these
infections.
Chemoprophylaxis with
ehrlichiosis in Africa.
tetracycline
for
canine
monocytic
B. Davoust1 , M. Boni1, J. Seignot1, D. Parzy2.
1Groupe de Secteurs Vétérinaires de Marseille, 2Institut de Médecine Tropicale du
Service de Santé des Armées, Marseille, France.
In the early eighties in French military kennels located in Africa there were many cases of
ehrlichiosis (CME) which were responsible for a variable intensity haemorrhagic syndrome . The
African kennels had lodged dogs permanently for several years and also dogs which came from
France for stays of about four months.
In the Dakar (Senegal) kennel, CME caused the death of half of the dogs (8/16) in 1987. Then
the prophylatic treatment (250 mg of tetracycline, per os, per day) was implemented. From 1987
to 1998, 177 dogs were treated during their stay. No toxic effects were observed, nor new
cases of morbidity and mortality caused by CME. All the systematic IFA tests realized on
treated dogs were negative.
In 1994 at the Abidjan (Ivory Coast) kennel, three of the six dogs tested were seropositive. In
1996, chemoprophylasis was implemented. The following years, no new serpositive case was
detected of 58 dogs.
In Djibouti, in the two military kennels set up successively in 1994 and 1995, the prevalence of
CME rose dramatically after new dogs arrived from France. In 1994, six out seven dogs were ill.
The fight against the vector was intensified and chemoprophylaxis put in place. Yet, from 1995
to 1998, 3 of 73 dogs monitored, were infected. Therefore the failure rate for this prophylaxy
was about 4 % in these kennels.
All things considered, 308 dogs were under chemoprophylaxis each day of their stay in Africa.
The estimated failure rate was about 0,9 % ( 3/308).
Total eradication of CME is not possible because of multiple interactions existing between
military dogs, ticks and local dogs.With regular administration of tetracycline, the selection of E.
canis antibioresitant strains is theoretically possible. For this reason, tetracycline is only
administered in an epidemiological context of major risk with medical supervision.
- 88 -
232C
Wednesday June 16th
Slide session 5
Rickettsioses in Animals, Hosts and Vectors
Validation and application of the Cowdria ruminantium-specific
pCS2O Tick PCR Assay
T.F. Peter1, B.H. Simbi1, A.F. Barbet2, R. Aileman2, M.J. Burridge2 and S.M.
Mahan"2
'UF/USAD/SADC Heartwater Research Project, Zimbabwe; 2University of Florida,
Gainesville, Florida. USA.
The development and use of the pCS20 tick PCR assay for Cowdria
ruminantium, the causative agent of heartwater disease of ruminants, has been
reported previously (Peter et al., 1995). Here, the assay was further evaluated in
laboratory and field studies on the Amblyomma tick vectors. The assay detected DNA
of 37 goegraphically diverse strains of C. ruminantium and had a specificity of 98%.
The assay did not detect DNA of the closely-related agent Ehrlichia chaffeensis.
Assay sensitivity varied with tick infection intensity and was high (97%) with ticks
bearing 107 organisms, but dropped to 28% with ticks bearing 102 organisms. When
applied to experimentally infected A. hebraeum ticks, PCR detected more infections
(93%) than the previons gold standard, the xenodiagnostic mouse inoculation assay
(8%) (kappa =0.015, p=0.17). The PCR assay was also applied to A. hebraeum and
A. variegatum field-co11ected ticks from 17 heartwater-endemic sites in four southern
African countries. Cowdria ruminantium infection was detected by PCR in all of the
tick batches, while attempts at tick transmission of infection to small ruminants failed
with four of the batches. The pCS20 PCR assay is presently the best characterized
and most reliable test for C. ruminantium in ticks and will be a valuable tool in
epidemiological investigations of heartwater.
- 89 -
12D
A Chemically Defined Medium for the in vitro Cultivation of Cowdria
ruminantium
43
Zweygarth, E. , A.I. Josemans and S.W. Vogel
Onderstepoort Veterinary Institute, Onderstepoort South Africa
The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in
domestic ruminants, was first achieved in 1985; since then, most groups working with
this culture system have used semi-defined media, which vary from batch to batch and
often fail to support the growth of C. ruminantium. We are therefore working towards the
replacement of serum-containing medium by a completely chemically defined medium for
Cowdria cultures. We attempted the long-term propagation of the Welgevonden stock of
C. ruminantium in bovine endothelial cell cultures in a variety of chemically defined
media. All media tested were based on Dulbecco's modified Eagle's medium nutrient
mixture Ham F-12 (DME/F-12), and RPMI 1640 with various supplements. One of the
media, referred to as SFMC-23, supported the long-term growth of the Welgevonden
stock of C. ruminantium for a total of 55 passages with regular passage intervals of 3
days. Other stocks of Cowdria ruminantium (Bloukrans, Mara, Sankat, Senegal) were
propagated for a test period of 10 passages with similar results.
Spotted Fever Group Rickettsiae in Ixodes ricinus ticks in Slovenia
T. Avsic-Zupanc, K. Prosenc, M. Petrovec
Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia
Ticks are known reservoirs and vectors of various pathogenic microorganisms.
Among them, Borrelia burgdorferi and TBE virus, are the most frequent ticktransmitted human pathogens in Central Europe. Ixodes ricinus represent the most
common tick species found in Slovenia, a country where Lyme borreliosis and tick
borne encephalitis are endemic. In order to investigate the possibility of the existence
of any other tick-borne microorganisms, ticks were collected from vegetation in five
different zoogeographical regions of Slovenia. A total of 1505 ticks I. ricinus collected
between 1996 and 1998 were examined for the presence rickettsiae by using
molecular techiques. DNA was extracted from every individual 490 adult ticks and
from 203 pools, each containing five nyphal ticks. By using PCR primers that amplify
a portion of citrate synthase-encoding gene (glta), specific sequences of the bacteria
belonging to the genus Rickettsia were detected on average in 11,16 % ticks. The
prevalence of infected ticks vary from 1,5 % found in Western Alpine region to 19,5
% detected in Prealpine region. RFLP analysis of the PCR amplicons revealed two
different restriction pattern. Phylogenetic analysis of the nucleotide sequences of the
two different samples showed that majority of the ticks (85 %) are infected with the
rickettsia closely related to R. helvetica. Whereas sequence analysis of twentytwo
samples conformed RFLP analysis which was similar to that seen in R. akari. These
preliminary data suggest that two SFG rickettsiae are present in I. ricinus ticks in
Slovenia with yet unknown pathogenicity to animals and humans.
- 90 -
79
The Many Faces of Wolbachia
Kostas Bourtzis1 and Henk R. Braig2
1 Insect Molecular Genetics Group, Institute of Molecular Biology and Biotechnology,
Foundation for Research and Technology - Hellas, Heraklion, Crete, Greece. - 2
School of Biological Sciences, University of Wales Bangor, Bangor LL57 2UW, U.K.
86
The intracellular endosymbiont Wolbachia pipientis, Rickettsiales, may be the
most ubiquitous intracellular bacterium as yet described in the animal kingdom. It has
so far been reported in numerous arthropod host species including isopod
crustaceans, mites and, in particular, insects. Current estimates indicate that from 15
to 30% of all insect species may carry Wolbachia. Its biology and phenomenology is
as wide ranging as its distribution.
It is well known to cause cytoplasmic
incompatibility in many insects, functional feminization of males in terrestrial isopods,
parthenogenesis in parasitic wasps and spider mites, sperm precedence and
increased male fertility in beetles and flies. Recent discoveries include the so-called
"popcorn" effect in Drosophila; the revelation that Wolbachia can parasitize on its own
phenotype; and the detection of Wolbachia in many species of filarial nematodes of
medical and veterinary importance. It is now also hypothesized that Wolbachia might
be responsible for the immunopathology of nematodes like Brugia malayi in humans
which would make Wolbachia a human pathogen. The future application of
Wolbachia ranges from use as a para-transformation tool for insects, as a spreading
force to replace natural insect populations of agricultural and public health importance
and as an organism that can change the senescence of disease vectors.
Strain structure of the ehrlichia Anaplasma marginale within
endemic herds and association with transmission.
T.F. McElwain, F.R. Rurangirwa, and G.H. Palmer.
Tick transmission of Anaplasma marginale in cattle occurs episodically and
unpredictably in endemic regions despite commingling of persistently infected and
uninfected cattle in the presence of vectors. Using retrospective analysis of A.
marginale isolates from outbreaks, individual strains can be identified using the msp1a
genotype, based on gene size and sequence. In experimentally infected animals, the
msp1a signature genotype remained stable with no nucleotide changes detected
through acute infection and at various time points during persistent infection. The
genotype was also stable during transmission using Dermacentor andersoni. No
nucleotide changes were present among samples taken from the blood of cattle at the
time of tick acquisition feeding, during development in the tick, and from the blood of
cattle following tick transmission. Thus, it appears that msp1a signature sequences
are stable within an outbreak strain. We determined the msp1a genotypes within a
closed, endemic herd of 300 cattle. Six different genotypes were present within this
closed herd and within an individual infected animal, each genotype was stable during
the 6-month period when tick-borne transmission was most likely. Transmission
within this endemic herd involved one of the strains present in the herd prior to
transmission, but did not represent a predominant genotype in the herd. In contrast to
the multiple genotypes present in an endemic herd, the signature msp1a genotype
from an acute outbreak in a herd from a non-endemic region was identical among all
10 animals involved. These findings suggest that transmission may be influenced by
ehrlichial genotype rather than simply being a stochastic event.
- 91 -
99
Rickettsia felis: Identification and molecular characterization in
fleas (Ctenocephalides felis) in Yucatán.
Jorge E. Zavala-Velázquez, José A. Ruiz-Sosa, Eric R. González-Sosa, and
David H. Walker.
Universidad Autónoma de Yucatán, Merida, Yucatán, Mexico and University of Texas
Medical Branch, Galveston, Texas
In 1996, for the first time the presence of antibodies to rickettsial antigens was reported in patients in Yucatán who
had a dengue-like clinical illness that was not dengue fever. Later studies revealed 5% prevalence of antibodies reactive
with R. akari in the population. However, the specific etiologic agent that stimulated these antibodies had not been
identified. In the present study a molecular characterization (PCR/RFLPs and sequencing) was performed to detect and
identify the prevalent rickettsial species in arthropods in this region.
The analysis was made on 1889 arthropods collected from 12 different towns in the State of Yucatán. The
identified species were Amblyomma cajennense, Riphicephalus sanguineus, Boophilus sp., Ctenocephalides felis and
Poliplax sp. Three different sets of conserved rickettsial primers were used to amplify specific sequences of the genes
encoding: 17 kDa, citrate synthase (RpCS.887p and RpCS1258n) and rOmpA (Rr190.79p and Rr190.602n). The amplified
products were detected in agarose gels stained with ethidium bromide. Alu I was used to digest the amplified product of glt
A, and Pst I and Rsa I to digest the amplified product of rompA. In addition, the amplified products of the 17 kDa and
citrate synthase genes were cloned into a pMOS vector and sequenced using commercial kits.
The PCR amplification showed that all the analyzed ticks and lice contained no detectable rickettsial DNA.
However, the fleas (C. felis) were positive. The RFLP's pattern of the glt A amplified product showed 3 single-T bands
(122, 87 and 81 bp) and a double-T band (43, 42 bp) similar to the reported SFG Alu I digest pattern. Additionally, the Pst I
RFLP presented 2 bands (404 and 106 bp), and the Rsa I RFLP revealed 2 bands (392 and 112 bp). Both patterns are
different from the previously reported bands for species within the SFG. The sequence analysis of the amplified 17 kDa
antigen gene revealed a 427 bp product; 100% homology with Rickettsia felis was observed for the 240 bp internal
sequence of the nested product. It also had the following homologies: R. rickettsii (93.9%), R. conorii (93.7%), R. typhi
(87.8%) and R. prowazekii (87.1%). Two sites for the Alu I restriction enzyme were recognized at the 127 and 236
positions of the sequence, similar to R. felis, R. rickettsii and R. conorii. The amplified product of the citrate synthase gene
presented a sequence of 381 bp; 100% homology with R. felis was observed for the 321 bp internal sequence of the
nested product. The following homologies were also observed: R. australis (96%), R. akari (96%), R. montana (96%), Thai
tick typhus rickettsia (96%), R. prowazekii (93%), and R. typhi (92%). Attempts to detect rickettsiae in blood samples from
dogs and other possible reservoirs in the area have not yielded positive results. Similarly attempted isolation of R. felis in
Vero cell cultures have not yet yielded rickettsial growth.
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147B
Wednesday June 16th
Poster session 6
Hosts, Vectors, Parasites and Taxonomy
Seroepidemiologic study of Rickettsia typhi infection in a rural
area of Spain
Lleda L., Saz W., Gegindez MI., Merino FJ., Beltran M.
Department of Microbiology and Parasitology, Faculty of Medicine, University of
Alcala, Spain
INTRODUCTION: Information about Rickettsia typhi infection is limited in
Spain. In general population has only been studied in a significant way in two
provinces: Salamanca and Sevilla. This work attempts to provide data for R. typhi
infection in the population from a rural area. So we selected the North of the
Autonomous Community of Madrid.
MATERIAL and METHODS: A total of 427 sera were researched for R. typhi
specific antibodies, belonging to the rural general population of 5 Health Area. It was
distributed proportionally by sexes (males -48%-, females -52%-), and geographical
areas according to information provided by the National Institute of Statistic.
Sera were examined by an indirect immunofluorescence assay (IFA) using
Vero E6 cells infected with R typhi (strain Wilmington). Anti-human total
immunoglobulins were used as FITC conjugated. Sera with antibodies titres > 1/40
were considered positive.
RESULTS: Specific R typhi antibodies were detected in 40 sera (9,3%).
Seroprevalence was 10,8% for women and 7,8% for men. The mean age among the
positives was 34,2 years with a standard deviation of 16,17 years. The percentages
observed of positives for geographical areas were: 8,1% for Paracuellos Jarama,
9,6% for Colmenar Viejo and 11% for La Cabrera.
CONCLUSION: The present survey proves the existence of R. typhi human
infection in a rural area of Madrid with a prevalence similar to that detected in
Salamanca (12,8%), and higher to that found in Sevilla (2%).
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11B
Molecular detection of Ehrlichia chaffeensis in ticks collected in
northwestern Florida.
1
2
17
1
John W. Sumner , Davis Janowski and Christopher D. Paddock .
1Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention,
Allanta, GA; 2 Florida Department of Health, Tallahassee, FL
Ehrlichia chaffeensis, an agent of human monocytic ehrlichiosis has recently
been isolated from six patients with ehrlichiosis from northern Florida. To investigate
the occurrence of E. chaffeensis in ticks in this region, questing ticks were flagged
from 6 sites in northwestern Florida in June1998. Specimens included 325 adult and
285 nymphal Amblyomma americanum, 11 adult Dermacentor variabilis, and 2 adult
Ixodes scapularis. DNA was extracted from individual adult ticks and from pooled
nymphal ticks (2 to 5 ticks per pool) and tested for the presence of the E. chaffeensis
variable length PCR target (VLPT) by using nested PCR. The VLPT contains 2 to 6
direct tandem repeat units and was targeted to investigate the geographic distribution
of size variants. Amplicons comigrating with E. chaffeensis VLPT markers were
detected in adult A. americanum collected from each of the sites and were amplified
from 4% to 41% of adult ticks collected at individual sites . E. chaffeensis was also
detected in nymphal A. americanum from 1 site, but not in D. variabilis from any site.
Four variants of the VLPT gene were found, containing 2, 3, 4 or 6 repeat units. Ticks
collected at three sites produced amplicons of a single or predominant variant, which
generally matched the VLPT profile observed in the human isolates acquired at the
same locations. Three different variants were amplified from tick populations at each
of the three other sites. We are currently sequencing the amplicons to confirm their
identity.
Detection of Ehrlichia Phagocytophila DNA in Ixodes ricinus from
areas in the north of Spain
Barral M; Benedicto L; Gil H; Juste RA; Garcia-Pérez AL
NEIKER (Instituto Vasco de Investigación y Desarrollo Agrarlo). 48160 Derio (Bizkaia)
Spain.
Ehrlichia phagocytophila is a rickettsia transmitted by Ixodes ricinus that causes
the so called tick-borne fever (TBF) in ruminants. This agent was reported in Spain in
cattle and it has been also implicated in a series of cases of enzootic abortions in
sheep in an area of the Basque Country. The purpose of the study was to determine
the prevalence of E. phagocytophila in free-living I. ricinus ticks and to stablish the
periods of the year where the risk of infection could be higher. A total of 4402 Ixodes
ricinus ticks (134 adults, 2011 nymphs and 2257 larvae) were collected from
vegetation along a complete year in 27 out of 37 geographic areas from the Basque
Country. DNA of Ehrlichia phagocytophila group was detected in adults and nymphs
in only those areas where a sufficient number of ticks were captured and processed.
30% of the areas presented positive results. 9.5% of adults and 1.6-13.7% of nymphs
were infected and infection was found along the year.
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21
Rickettsia-like organisms in ticks (acari: Ixodidae) in South Africa
A-M Pretorius 1, GHJ Pretorius 2, LJ Fourie 3 & PJ Kelly 4
Departments of Medical Microbiology1, Haematology2, Faculty of Health Sciences,
Department of Zoology/Entomology3, Faculty of Science, UOFS, BLOEMFONTEIN,
South Africa, Faculty of Veterinary Sciences4, University of Zimbabwe, Harare,
Zimbabwe
77C
The spotted fever group (SFG) of rickettsiae are maintained in ticks and their
mammalian hosts. Rhipicephalus sanguineus is recognised as the major vector of
Rickettsia conorii in Europe. Previous studies on southern African ticks have shown
that various species are infected with RLOs, mainly Amblyomma hebraheum (72%),
A. sparsum (33%), A. variegatum (17%), Hyalomma marginatum rufipes (11%), R.
simus (11%), Haemaphysalis leachi (6%), A. rhinocerotis (5%) and H. truncatum
(5%).
Hard-bodied ticks were collected from domestic and wild animals in the Free
State, Northern Cape, Eastern Cape and Kwazulu/Natal provinces of South Africa.
The prevalence of rickettsia-like organisms (RLOs) in ticks was determined by using
haemolymph testing. Tick species that were infected with RLOs included Amblyomma
hebraeum (40/57; 70%) , Hyalomma marginatum rufipes (40/110; 36%), H. truncatum
(32/84; 38%), Boophilus decoloratus (23/63; 37%), Haemaphysalis leachi (13/46;
28%), H. silacea (4/7; 57%), R. evertsii (39/113; 35%), R. appendiculatus (15/15;
100%), R. distinctus (1/1; 100%), R. mühlensi (11/13; 85%), R. punctatus (1/2; 50%),
and R. simus (1/2; 50%). No RLOs could be demonstrated in R. sanguineus (0/331),
and A. marmoreum (0/1).
This study indicates that in South Africa RLOs may be found in a large number of tick
species, many of which have not been previously described as being infected with
these organisms in Africa. Further studies, mainly PCR-RFLP, are indicated to
characterize the RLOs in ticks from South Africa
Prevalence of Bartonella henselae in pet cats in the United States
L Guptill1, C-C Wu1, L Slater2, L Glickman1, M. Faderan1, H Syme1, H HogenEsch1
1Purdue University, W. Lafayette, IN 47907
2University of Oklahoma, VA Medical Center, Oklahoma City, OK 73104
Zoonotic Bartonella henselae (Bh) infection causes cat scratch disease (CSD),
bacillary angiomatosis, and other human illnesses. Domestic cats are the major
reservoir and vector for human infection.
Closely related to Bh, Bartonella
clarridgeiae (Bc) also infects cats, and may be a rare cause of CSD. In past studies
up to 50 % of stray cats or cats in shelters were antibody- or blood culture-positive for
Bh. However, the prevalence of Bh bacteremia in household cats has not been
evaluated systematically, despite the fact that these cats are probably the source of
most human infections. Consequently, the purpose of this study was to determine the
prevalence of Bartonella bacteremia and serum antibodies in pet cats <2 years old
seen at private veterinary practices in 1997 and 1998 in 4 regions of the US. Blood
culture results were positive for Bh (speciated by immunofluorescence) in 33% of cats
examined in Florida, 28% of cats in California, 12% of cats in the Washington, DC
area, and 6% of cats in the Chicago, IL area. Anti-Bh antibodies (detected by enzyme
immunoassays) were present more often in cats from Florida (68.1%) and California
(62.9%) than in Washington DC (28%) or Chicago (12.5%). In contrast to the regional
distribution of Bh seroprevalence, a larger proportion of cats in Washington, DC
(12.5%) and Chicago (10.4%) had positive Bc titers than in Florida (5.3%) or
California (5.2%). No blood cultures in this study yielded Bc.
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81
Partial citrate synthase gene sequences of Bartonella species
detected in rodents and sand flies in Caraz, Peru
1
1
1
115
2
Scott W. Gordon , Richard G. Andre , W. Patrick Carney , Eric L. Marston , Guy
Hohenhaus1, Roberto Fernandez3, Larry W. Laughlin1, Douglas M. Watts3, and
Russ Regnery2
1 Department of Preventive Medicine and Biometrics, Uniformed Services University
of the Health Sciences; 2 Division of Viral and Rickettsial Diseases, CDC, Atlanta; 3
Naval Medical Research Institute Detachment, Peru,
Bartonellosis is a potentially life threatening disease which occurs in the Andes
Mountains of South America. The disease is caused by the bacteria, Bartonella bacilliformis,
and is believed to be transmitted to humans through the bite of an infected Lutzomyia sand fly.
To better understand the epidemiology of endemic bartonellosis in the Chavin Region of Peru,
we examined wild rodents and sand flies by culture and PCR for evidence of infection with the
bacteria.
Sand flies and rodents were collected in and around the residences of patients with
Bartonellosis in the Caraz Valley, Chavin Region, Peru. Total DNA was extracted from sand
flies and rodent blood using a commercial isolation kit (Qiagen). Rodent blood samples were
also cultured on blood agar plates. Bacterial DNA isolated from positive cultures was prepared
as described above. DNA preparations were screened for presence of Bartonella species using
PCR primers for Bartonella henselae described by Norman et al., (1995) and positive products
were sequenced.
Six distinct genotypes, including B. bacilliformis, were detected in 30 of 152 sand fly
pools (28 Lutzomyia verrucarum and 2 L. noguchi) collected during 1997 and 1998. Nine
distinct Bartonella genotypes were recovered from 32 of 602 rodents (primarily Mus musculus,
Akodon mollis, Oryzomys zantheolus, and Phyllotis andium). These results indicate that
multiple genotypes of Bartonella are circulating in rodent and sand fly populations in an area
endemic for human bartonellosis. The detection of B. bacilliformis in L. verrucarum provides
support for the hypothesis that this sand fly can serve as a human vector of the disease.
Molecular detection of Borrelia and Ehrlichia in tick samples of a
North-Eastern Italian province
1
G.Favia, 1G.Cancrini, 2E.Lillini, 2G.Macrì, 1A.Carfì, 3D.Grazioli, 4E.Gatti, 1A.Iori.
Istituto di Parassitologia Università "La Sapienza", Roma, Italy - 2 Istituto
Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Roma, Italy - 3
Dipartimento Prevenzione ULSS 2-Feltre (BL), Italy - 4 AS. F.D. - Belluno, Italy
1
Borrelia burgorferi s.l. has been isolated in invertebrate and vertebrate hosts in
Northern and Central Italy where several cases of Lyme Diseases (LD) have been
also reported. Ehrlichia phagocytophila has been isolated in Ixodes ricinus collected
in the protected area of Manziana (Central Italy), but no clinical cases of Human
Granulocytic Ehrlichiosis (HGE) have been reported in the country.
The aim of this study was to detect by molecular diagnostics (PCR)
B.burgdorferi s.l. and Ehrlichia in I.ricinus collected in Feltre district (Belluno province,
NE Italy) where LD is present and Ehrlichia seropositivity has been found in high-risk
subjects.
The first sample of I.ricinus nymphs (N=55), caught by dragging four areas of
Feltre district, was divided in 11 pools and DNA extraction was performed. DNAs
were used as template in separate PCRs with specific primers for Borrelia and for
Ehrlichia. Six pools were negative, one was positive for both pathogens and four
pools only for Borrelia. The first pool was then tested in a single PCR with both pairs
of primers to evaluate the chance to detect both pathogens simultaneously, and the
results were consistent.
The preliminary findings document the presence of B.burgdorferi s.l. and
Ehrlichia spp in the alpine area of Belluno province. The usefulness of PCR in the
detection of these micro-organisms in areas of particular interest is confirmed.
Moreover the development of a single PCR assay, that may detect both parasites all
at once, would allow much saving of time and costs.
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117
Investigation of natural foci with spotted fever in Eastern of
Guangdong Province
132D
Zhang Jian-zhi 1,Bi de-zeng 1,He Jin-rong 1,Chen Min 1,Gou Yan 2,Pan Linxiang3, Luo Jing-ping 4
1 Institute of Epidemiology and Microbiology£¬Chinese Academy of Preventive
Medicine£¬Beijing 102206 - 2 Shantou Medical College, Shantou University, Shantou
515031 - 3 Meizhou City Sanitary and Anti-epidemic Station,Meizhou 514000 - 4
Dapu County Sanitary and Anti-epidemic Station,Huliao 514200
Investigation of natural foci with spotted fever were performed in Dapu county
of Guangdong Province. 282 healthy persons, 53 wild mice,30 cattle,2 sheep and 494
ticks were investigated.The results show that the major wild mice are Rattus
norvegicus and R.losea in Dapu county ;the tick-bearing rate were respectively
15.09%(8/35),100%(35/35) and 100%(2/2) in wild mice,sheep and cattle;the morbidity
rate of mice was 49.09% (26/53); the major ticks were Haemaphysalis cornigera
taiwana, Dermacentor auratus and H.hystricis; the prevalence of antibodies to
R.sibirica, R.conorii and R.akari were respectively 10.28% (29/282), 3.90% (11/282)
and 5.67%(16/282)among healthy persons; respectively 13.33%(4/30), 3.33%(1/30)
and 10.00%(3/30) in wild mice and respectively 8.00%(2/25),4.00%(1/25) and
4.00%(1/25) in cattle.
An attempt for monitoring of tick spotted fevers in Bulgaria
E. Aleksandrov 1,2, D. Mitov1, D. Aleksandrova 1,2, B. Kamarinchev 1,2, N.
Bogdanov1.
Military Medical Academy - 1; National Referent Laboratory for Rickettsioses - 2
Cases of Mediterranean Spotted Fever were not registered in Bulgaria for more
than 20 years. After 1994 such cases have appeared again and their number is
increasing in the last years. That new wave of the endemic for the region rickettsiosis
is characterized by some peculiarities in the clinical course and the epidemiology of
the disease.Particular interest presents the differences in the clinical course in the
different regions.A study of the antibody titers against the main representatives of the
spotted fever group /SFG/ rickettsiae: R. conori, R. sibirica, R. rickettsii has been
carried out with the help of micro-immunofluorescent test. It was found out that the
highest titers of antibodies against R. conori in the examined cases were 22.56%,
against R.sibirica 26.66%, against R.rickettsii - 14.87%. With equal titers against
R.conori and R.sibirica reacted 13.85%, against R.conori and R.rickettsii - 9.7%,
R.sibirica and R.rickettsii 4.62%, and antibodies against the three rickettsioses were
established in 7.69%. Antibodies against R.rickettsii prevailed in the regions
characterized with high incidence and heavier clinical course. For all other regions the
percentage of sick persons with antibodies against R. rickettsii was 3-4 fold lower or
antibodies of the same kind were not found but antibodies against R. conori and R.
sibirica dominated. In part of the sera antibodies against 2 or 3 antigens were
established.The received results correspond with data from previous studies which
had found out that different rickettsioses had been circulating in the territory of
Bulgaria /the studies were carried out with species- and group-specific monoclonal
antibodies/.The monitoring of SFG rickettsioses in Bulgaria has theoretical and
practical value in the effective measures against them.
- 97 -
138C
Isolation of Rickettsia conorii strain from
sanguineus ticks collected from dogs in Egypt
Rhipecephalus
139
Ashraf G. El Dessouky , Andrew J. main and Imam Z. Imam,
Ain shams University, Research and Training Center on Vectors of
Diseases,Abbassia, Cairo, Egypt; Biology Department, American University of
Cairo;United States Naval Medical Research Unit No. 3 (NAMRU-3), Egypt
A field study was initiated in 1992 and continued for two successive years to
attempt the isolation of rickettsial species belonging to the Spotted Fever Group
(SFG) which is circulating in Egypt without being isolated before, and to investigate
preliminary whether the Egyptian strain of SFG is similar or not to the Israeli strain. A
total number of 224 dogs within the Metropolitan Cairo and Belbeis province (Sharqiya
Governorate) were screened for the presence of ticks. A total of 1021 Rhipecephalus
sanguineus ticks and a total number of 144 rodents were captured and screened. Of
the 1021 tick hemolymphs examined, 13 contained immunofluorescent rickettsiae. All
the Hemolymph test positive ticks were subjected to isolaton attempts using the shell
vial technique and inoculation in male guinea pigs. The isolated strain was identified
by the Fluorescent Antibody technique and confirmed by conducting electrophoretic
analysis of protein antigens (SDS-PAGE and western immunoblotting). The study
revealed the presence of a circulating strain (Egyptian Strain) belonging to R. conorii,
a member of SFG rickettsiae, and that this strain is characterized by being avirulent to
both animals ( i.e. did not induce symptomatic infection in guinea pigs and mice) and
cell cultured cell lines ( i.e. CPE could not be detected in the infected cell lines).
Studies on detection of Rickettsia tsutsugamushi dna in the single
larva of Leptotrombidum scutellare collected from the endemic
areas by PCR
Guo Hengbin et al .
Institute of Military Medicine,Nanjing Command,PLA,Nanjing 210002
Nested polymerase chain reaction (NPCR) was used to detect Rickettsia
tsutsugamushi (R,t) DNA in the single larva of L.(L.) scutellare collected from the
endemic areas of tsutsugmushi disease in Jiangsu province. The primer pairs for PCR
were designed on the basis of the nucleotide sequence of the gane that was encoded
the 56-KDa antigen. R.t specific DNA band of 481-507bp,were observed by NPCR in
2 of the DNA estracted from 61 larvae. The results proved that these two larvae of
L.(L.) scutellare carried Rt and Rt carrying rate was 3.27%.
The results indicated that this method may be used to detect Rt in the sing
lauva of chigger mite, and is possessed of practical value in the eqidemioligical
investigation of vector mites of tsutsugamushi desease in an area.
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165C
Prevalences of selected bacterial pathogens in adult Ixodes
scapularis from the middle atlantic region of the United States
173
Joseph E. Bunnell, Timothy M. Shields, and J. Stephen Dumler
Department of Molecular Microbiology and Immunology , The Johns Hopkins
University School of Hygiene and Public Health
An emerging tick-borne infectious disease of North America and Europe,
human granulocytic ehrlichiosis (HGE) is a potentially fatal undifferentiated febrile
illness with geographic distribution and epidemiology largely overlapping those of
Lyme disease. In the Middle Atlantic region of the US, the etiologic agents for both of
these human pathogens are vectored by Ixodes scapularis. Preliminary studies reveal
tick infection prevalences different than those predicted based on rates in the
Northeast and Upper Midwest. Adult I. scapularis were collected in a systematic
manner from 17 locations in the Middle Atlantic Region of the US. Within each of the
17 locations, 7 sites of 200 m transects (15 mins./site; 1.75 hrs./location) were
sampled. Ticks were assayed for infection with the HGE agent, Borrelia burgdorferi,
and Wolbachia pipientis by PCR for the 16S rRNA, OspA, and Wosp outer surface
protein genes, respectively. Tick abundance patterns were highly clustered, with
relatively high numbers along the coastal plain of the Chesapeake Bay, decreasing to
the west and south. Overall, from all ticks collected in this region of the country, HGE
agent and B. burgdorferi infection prevalence rates are lower than reported in other
parts of the US (<1% and 20%, respectively). Environmental variables were examined
for correlation to tick infestation and infection patterns. A computer model which can
be validated by ground-truthing is being developed, enabling predictions of infected
tick abundance patterns.
Transmission of granulocytic Ehrlichia from the tick vector, Ixodes
scapularis , to the mouse host, Peromyscus leucopus
Douglas, J., N. Miller, MJ Mauel, TN Mather.
Center for Vector-Borne Disease, University of Rhode Island, Kingston, RI, USA
The purpose of this study was to determine the nymphal tick attachment time
necessary before transmission of Granulocytic Ehrlichia (GE). Nymphal Ixodes
scapularis ticks, infected as larvae were allowed to fed on uninfected Peromyscus
leucopus mice for various time intervals of 6, 12, 18, 24, 48 hours and to repletion.
The time necessary for the transfer of infection to the mice was determined to be 6
hours. This has Public Health implications by greatly reducing the time allowed from
tick attachment to tick removal before transmission of tick-borne infection.
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176
Characterization of a new Spotted fever group rickettsia isolated
from Ixodes ricinus ticks collected in Slovakia
1,2
1
2
2
191A
1
Z. Sekeyova , V. Roux , E. Kocianova , J. Rehacek , D. Raoult
1Unité des Rickettsies, Faculté de Médecine, CNRS UPRES-A 6020, 13385 Marseille,
France. 2Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 842
46 Bratislava, Slovakia.
A new strain of rickettsia was detected from two individual Ixodes ricinus ticks
by PCR. No. 3 was DNA extracted from tick collected in Northeastern Slovakia and
No. 4 from tick collected in Southwestern Slovakia. Sequence determination of 16S
ribosomal DNA (16S rDNA), citrate synthase gene (gltA) and rOmpA outer membrane
protein encoding gene (ompA), showed genetical similarity of the two new rickettsiae
which diverge from identified Spotted fever group rickettsial genotypes.
Phylogenetical analysis inferred from gltA and 16S rDNA sequence comparison
showed that this newly recognized rickettsial genotype is most closely related to
Rickettsia helvetica, whereas the one inferred from ompA sequence clustered No. 3
and No. 4 with Rickettsia montana in the Rickettsia massiliae subgroup (sequencing
of Rickettsia helvetica ompA has not been done). Significant boostrap values for the
nodes where branched No. 3 and No. 4, in the dendrograms inferred from gltA and
ompA sequence comparison, were obtained. These data suggest that this newly
recognized rickettsia should be considered a distinct taxonomic entity.
Phylogenetic analysis of the bacteria included in the genus
Rickettsia using sequence comparison of the gene coding for a
120 kD protein in Rickettsia conorii.
Z. Sekeyova, V. Roux, D. Raoult.
Unité des Rickettsies, Faculté de Médecine, CNRS UPRES-A 6020, 13385 Marseille,
France.
Using PCR and an automated DNA sequencer, we amplified and sequenced a
3510 bp fragment of the gene coding for a 120 kDa protein in Rickettsia conorii, in 26
representatives of the genus Rickettsia. Phylogenetic analysis inferred from sequence
comparison defined a large subgroup including Astrakhan fever rickettsia, Israeli tick
typhus rickettsia, Indian tick typhus rickettsia, Rickettsia conorii strain moroccan,
Rickettsia africae, strain S, «Rickettsia mongolotimonae», Rickettsia sibirica and
Rickettsia parkeri. A second smaller subgroup included Bar 29, Rickettsia massiliae
and Rickettsia rhipicephali. Compared with the previous phylogenetic reconstructions,
the position of R. aeschlimannii and Rickettsia montanensis outside of this group is
unexpected. Rickettsia australis, Rickettsia helvetica and a new isolate No. 4
diverged first from the common rickettsial ancestor. When distance matrix and
parsimony methods were used to construct the dendrogram, the same general
organization was obtained, excepting the position of Rickettsia rickettsii. We were not
able to amplify this gene for Rickettsia canadensis and Rickettsia bellii. Sequence
comparison of this gene could be a complementary approach to infer rickettsial
evolution as significant boostrap values were obtained for most of the nodes.
- 100 -
191B
Interactions between Wolbachia and their drosophila hosts
Stephen Hadfield
Department of Zoology Oxford University
Wolbachia are a genus of maternally inherited endosymbiotic bacteria,found
commonly in the reproductive tissues of arthropods. Closely related to the Rickettsial
typhus agents, they are responsible for various phenomena including; cytoplasmic
incompatability, parthenogenesis and feminization of genetic males. These phenotypic
effects all manipulate the hosts' reproductive systems in order to increase the number
of infected individuals within the population. Wolbachia present a fascinating
spectrum of research possibilities from modelling speciation to controlling insect born
diseases such as African trypanosomiasis, by engineering symbionts carrying genes
capable of disrupting trypanosome transmission. Since they are known to be largely
vertically transmitted, we might predict the existence of an evolutionary pressure
selecting for bacteria which become localised to the host germ cells during
development.Using newly developed techniques in multi-photon electron microscopy,
we have shown that Wolbachia become concentrated at the site of germ cell
precursor development at the posterior pole of the Drosophila embryo. We then used
strains of Drosophila melanogaster bearing mutations in posterior patterning genes,
to demonstrate that this localisation is dependant upon some genetic factor
associated with germ plasm assembly.
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256